Ellen Holm Nielsen

Only amyloidogenic intermediates of transthyretin induce apoptosis

In diseases like Alzheimers disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

Electron microscopy of prefibrillar structures and amyloid fibrils.

Publisher Summary Several techniques, such as X-ray crystallography, light scattering, fluorescence spectrometry, size exclusion chromatography, atomic force microscopy, and transmission electron microscopy, have been employed in studies of structural intermediates of fibril formation and fibrillar assembly of amyloid proteins. Electron microscopy, with a resolution of approximately 2 nm, offers a useful technique for the ultrastructural characterization of preprotofilaments, protofilaments, and mature fibrils formed during in vitro fibrillogenesis. For contrast enhancement of specimens, negative staining is applied. This chapter outlines the methodology used in laboratories for electron microscopic examination of negatively stained prefibrillar structures and amyloid fibrils. The A β -peptide used influences the kinetics of the fibril formation. This chapter uses A β 1–42 (Bachem, Bubendorf, Switzerland), which forms fibrils within a few hours of incubation at 37°. In order to decelerate the fibril formation enabling it to investigate early intermediates and prefibrillar structures, this chapter has performed the in vitro studies at low concentrations of A β 1–42 (170 μg/ml), as the kinetics of fibril formation is highly concentration dependent.

Electron microscopic study of the regeneration in vitro of rat peritoneal mast cells after histamine secretion

SummaryRegeneration of rat mast cells was studied by TEM from 10 s to 48 h after secretion of histamine induced by compound 48/80. During the first 2 h, small intracellular cavities, formed during compound exocytosis and containing non-membrane-bound remnants of the granules, tended to coalesce, and after 2 h of incubation regeneration started. After 6 h, all the cavities had fused into one large central cavity which contained the remnants of the granules and remained open to the exterior during the entire period. The plasma membrane microfolds which disappeared just after secretion were reformed during regeneration. They were apparently involved in endocytotic-like activity and coated vesicles also appeared beneath the plasmalemma (membrane recycling?). The fate of the granule remnants in the cavity is unknown, as regeneration was not completed after 48 h which is the longest survival time obtained so far in ultrastructural studies of mast cell regeneration in vitro.

Increased plasma concentration of serum amyloid P component in centenarians with impaired cognitive performance

Serum amyloid P component (SAP) binds to all amyloid fibrils including those in the plaques and tangles of Alzheimer patients. To investigate whether the plasma SAP concentration correlated to cognitive impairment, we measured SAP levels in blood samples from 41 centenarians and compared these to the cognitive performance evaluated by Mini Mental State Examination (MMSE). We observed a significantly (p < 0.001) increased SAP concentration (48.3 ± 16.9 µg/ml; mean ± SD) in the centenarians compared to gender-matched controls (32.8 ± 11.4 µg/ml). Six severely demented centenarians had an even higher SAP concentration (60.2 µg/ml), while the subgroup of cognitive intact centenarians (MMSE score >24) showed a normal SAP concentration (38.4 ± 9.3 µg/ml). No dehydration or hepatic dysfunction was demonstrable in the centenarians. We conclude that the centenarians with impaired cognitive performance had significantly increased plasma concentrations of SAP, while the values for cognitive intact centenarians were within the normal range.

Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins: enhanced binding at slightly acid pH.

Serum amyloid P component (SAP), a member of the pentraxin family of proteins, binds calcium-dependently to several ligands including glycosaminoglycans (GAGs). We have investigated the influence of pH on the Ca2(+)-dependent binding of SAP to solid phase GAGs and amyloid fibril proteins (AA and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under conditions of slightly lowered pH.

Calmodulin in mast cells and its role in histamine secretion

Calmodulin content and distribution in rat peritoneal mast cells was determined by radioimmunoassay. Isolated pure mast cells were disrupted by sonication and the total calmodulin content and its distribution were determined. Calmodulin bound to the membranes was released by heating with 0.1% Lubrol PX to 95°C for 5 min. The total calmodulin content of mast cells was found to be 160±14 ng/106 cells (9.4±0.82 pmoles/106 cells). The highest amount (68%) was present in the supernatant representing the cytosol. The next highest amount (26%) was found in the composite fraction consisting of mitochondria, endoplasmic reticulum, Golgi vesicles and plasma membrane (100,000g pellet). The mast cell granules contained 4% of the total calmodulin.Trifluoperazine (TFP) was used as an antagonist to explore the role of calmodulin in histamine secretion. At 10 μM concentration, TFP caused a negligible spontaneous histamine release by its membrane effect. TFP (10 μM) inhibited histamine release by all the three secretagogues used, but the degree of inhibition varied: 60% with antigen, 40% with compound 48/80 and 20% with ionophore A23187. It is suggested that the TFP effect is due to calmodulin-antagonism and interference with the activation of enzymes, essential to the secretory process.

Cytoskeletal studies on lowicryl K4M embedded and affi-gel 731 attached rat peritoneal mast cells

SummaryThe subplasmalemmal network in mast cells consists of irregularly arranged 6–7 nm filaments (actin) connected by thinner filaments. In places oblique filaments with crossbridges or short, perpendicular filaments (11–12 nm) connect cell and granule membrane. Filaments attaching subplasmalemmal network to cell membrane divide like a Y and attach cell membrane end-on with a conical, hook-like bending. Each granule is surrounded by a regular network of filaments.

Complexes of serum amyloid P component and DNA in serum from healthy individuals and systemic lupus erythematosus patients.

Serum amyloid P component (SAP) bindsin vitro to DNA; based on findings in SAP-deficient mice it was proposed that SAPs role is to handle chromatin and DNA, thereby preventing formation of anti-DNA antibodies. For the first time we have shown the presence of Ca2+-dependent SAP-DNA complexes, measured by ELISA, in sera from both healthy volunteers and systemic lupus erythematosus patients (SLE). The concentration of SAP-DNA complexes in SLE sera was significantly lower than in normal sera and particularly low in sera from patients with anti-DNA titers exceeding 50. The complexes were dissociated by the SAP ligand heparin and were not demonstrable in EDTA plasma.Normal sera showed similar capacity to form SAP-DNA complexes with both thymus andEscherichia coli DNA, whereas significantly lower amounts of complexes, in particular withE. coli DNA, were formed in SLE sera. SLE patients with moderate to high anti-DNA titers showed a significant negative correlation between serum SAPs binding ofE. coli DNA and the anti-DNA titer.

The involvement of protein kinase C in exocytosis in mast cells

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.

Surface morphology of rat peritoneal mast cells during in vitro regeneration after histamine secretion

SummaryCell-surface morphology of regenerating mast cells was followed over a period of 48 h after histamine release. Control cells (not stimulated to secrete) were characterized by anastomosing folds of membrane of equal depth and width. During exocytosis these folds disappeared and were replaced by deep cup-shaped flaps of membrane evident in cells incubated for 10 min. During the first hours of regeneration these flaps fused mutually or with the plasma membrane. This activity suggests membrane retrieval, maybe specifically recycling the granule-type patches of membrane. Membrane-fusion activity was observed to some degree also after extended incubation. After 48 h of incubation the regeneration process was still not completed, as indicated by the fact that holes leading to intracellular cavities could still be found.