DNA aptamer raised against advanced glycation end products (AGEs) improves glycemic control and decreases adipocyte size in fructose-fed rats by suppressing AGE-RAGE axis.

Advanced glycation end products (AGEs) decrease adiponectin expression and suppress insulin signaling in cultured adipocytes through the interaction with a receptor for AGEs (RAGE) via oxidative stress generation. We have recently found that high-affinity DNA aptamer directed against AGE (AGE-aptamer) prevents the progression of experimental diabetic nephropathy by blocking the harmful actions of AGEs in the kidney. This study examined the effects of AGE-aptamer on adipocyte remodeling, AGE-RAGE-oxidative stress axis, and adiponectin expression in fructose-fed rats. Although AGE-aptamer treatment by an osmotic mini pump for 8 weeks did not affect serum insulin levels, it significantly decreased average fasting blood glucose and had a tendency to inhibit body weight gain in fructose-fed rats. Furthermore, AGE-aptamer significantly suppressed the increase in adipocyte size and prevented the elevation in AGEs, RAGE, and an oxidative stress marker, 8-hydroxydeoxyguanosine (8-OHdG), levels in adipose tissues of fructose-fed rats at 14-week-old, while it restored the decrease in adiponectin mRNA levels. Our present study suggests that AGE-aptamer could improve glycemic control and prevent adipocyte remodeling in fructose-fed rats partly by suppressing the AGE-RAGE-mediated oxidative stress generation. AGE-aptamer might be a novel therapeutic strategy for fructose-induced metabolic derangements.

Bazedoxifene blocks AGEs-RAGE-induced superoxide generation and MCP-1 level in endothelial cells.

Advanced glycation endproducts (AGEs) and their receptor (RAGE) play a role in vascular complications in diabetes. We have previously shown that 17ß-estradiol at 10 nmol/l, a nearly identical plasma concentration to that during mid-pregnancy, up-regulates RAGE expression in endothelial cells. The finding might suggest the involvement of 17ß-estradiol in the deterioration of vascular complications in diabetes during pregnancy. However, the effects of the selective estrogen receptor modulator, bazedoxifene, on oxidative and inflammatory reactions in AGEs-exposed endothelial cells remain unknown. In this study, we addressed the issue. Ten nmol/l 17ß-estradiol increased RAGE and monocyte chemoattractant protein-1 (MCP-1) gene and protein expression in human umbilical vein endothelial cells (HUVECs), both of which were blocked by 10 nmol/l bazedoxifene. Bazedoxifene at 10 nmol/l also significantly inhibited the AGEs-induced superoxide generation, RAGE and MCP-1 gene and protein expression in HUVECs. The present study suggests that blockade of the AGEs-RAGE axis by bazedoxifene might be a novel therapeutic target for preventing vascular damage in diabetes, especially in postmenopausal women.

Iron lamination and interlaminar insulation for high-frequency pulsed magnets.

In high-frequency pulsed magnets, such as kickers in particle accelerators, it is essential to reduce eddy currents that could be induced in the magnet core during excitation not to distort and attenuate the magnetic field pulse. A novel iron lamination scheme with additional interlaminar insulation is proposed for the magnet core of such pulsed magnets. A laminated steel sheet core is formed by alternately stacking thin steel and insulation sheets. For application to matched kicker magnets for accelerators, test magnets with the new and conventional iron lamination were designed, assembled, and extensively evaluated. The pulsed magnetic field waveforms of two test magnets with the new lamination successfully matched to below 0.1% over the entire pulse duration, which was significantly better than those with the conventional lamination. Among the applications of the developed high-frequency pulsed magnets, beam injection kickers for the coming next generation light sources and future colliders, where suppression of the transient stored-beam oscillation during beam injection is crucial, are considered to be promising.

Large-scale flooding analysis in the suburbs of Tokyo Metropolis caused by levee breach of the Tone River using a 2D hydrodynamic model.

In order to assess the effects of climate change on flood disasters in urban areas, we applied a two dimensional finite element hydrodynamic model (2D-FEM) to simulate flood processes for the case analysis of levee breach caused by Kathleen Typhoon on 16 September 1947 in Kurihashi reach of Tone River, upstream of Tokyo area. The purpose is to use the model to simulate flood inundation processes under the present topography and land-use conditions with impending extreme flood scenarios due to climate change for mega-urban areas like Tokyo. Simulation used 100 m resolution topographic data (in PWRI), which was derived from original LiDAR (Light Detection and Ranging) data, and levee breach hydrographic data in 1947. In this paper, we will describe the application of the model with calibration approach and techniques when applying for such fine spatial resolution in urban environments. The fine unstructured triangular FEM mesh of the model appeared to be the most capable of introducing of constructions like roads/levees in simulations. Model results can be used to generate flood mapping, subsequently uploaded to Google Earth interface, making the modeling and presentation process much comprehensible to the general public.

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres.

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.

A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42.

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism.

The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P(2), and promote its retention and clustering. Binding and modulation of PI(4, 5)P(2) depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P(2) modulation. In the neuron-like cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by DeltaED mutants. Agents that globally mask PI(4,5)P(2) mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(DeltaED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P(2) modulating proteins, upstream of actin and cell cortex dynamics regulation.

Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP2.

The turnover of phosphatidylinositol 4,5-bisphosphate (PIP2) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including mitogens, but decisive evidence for the idea has not been obtained. In the present study, a monoclonal antibody to PIP2 was microinjected into the cytoplasm of NIH 3T3 cells before or after exposure to mitogens. The antibody completely abolished nuclear labeling with [3H]thymidine induced by platelet-derived growth factor and bombesin, but not by fibroblast growth factor, epidermal growth factor, insulin, or serum. The findings strongly suggest that PIP2 breakdown is crucial in the elicitation and sustaining of cell proliferation induced by some types of mitogens such as platelet-derived growth factor and bombesin.

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction.

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.

Performance verification of a precise vibrating-wire magnet alignment technique for next-generation light sources.

The high-accuracy alignment of magnets is a key issue in the development of next-generation light-source rings. To obtain adequate dynamic apertures, the magnets must be aligned to an accuracy of 10 µm or better. Recently, a new technique that utilizes a vibrating wire has attracted attention for this purpose as it can directly determine with high resolution the magnetic centers in a series of multipole magnets on a straight section between bending magnets. In conventional vibrating-wire alignment techniques, wire sag, which causes alignment errors, is determined from the theoretical catenary curve. By contrast, in the present study, we have measured the sag profiles of various wires in the longitudinal direction to micrometer-order accuracy. We concluded that we can reduce deviations of the actual wire sag from the theoretical curve by choosing a suitable wire. By setting up a test bench of a vibrating-wire alignment system for a series of multipole magnet on a straight section, we have achieved the total error of the magnetic-center measurements of micrometer-order in the standard deviation. Moreover, two systematic error factors, the drift of the magnetic centers due to thermal deformations of the magnets after they are excited and the change in the magnetic centers due to reassembly of the magnets after installing the vacuum chamber, are included in practical magnet alignments. We have experimentally investigated these error factors using the test bench.

Rejection of pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs) by low pressure reverse osmosis membranes.

This paper aims to elucidate retention characteristics of some pharmaceuticals and personal care products (PPCPs), and endocrine disrupting chemicals (EDCs), by two polyamide low pressure reverse osmosis (LPRO) membranes. Feed solution pH did not have an influence on rejections of undissociated solutes, which was most likely governed by adsorption, size exclusion and diffusion simultaneously. Size exclusion was presumably dominant, especially with tight membranes (UTC-70U). Rejections of the solutes with low dipole moment (<1.0 debye) decreased with increasing octanol-water partition coefficient (K(ow)). The solutes with large K(ow) values were most likely adsorbed on membrane and subsequently passed through it resulting in larger diffusion coefficient (D(p)). The rejections decreased with increasing D(p) values irrespective of their dipole moments. Rejections of solutes with comparatively larger dipole moments might be dominated by diffusion and/or convection rather than their hydrophobicity. However, rejections of solutes with hydroxyl and carboxyl functional groups by UTC-60 increased with solution pH. More than 80% rejections were obtained for degree of dissociation (alpha)>0.5. Electrostatic repulsion played a key role for rejection of dissociated solutes, especially by loose LPRO membranes. Therefore, assessing the dissociation degree at desired pH values can be a key step to obtain an insight of rejection mechanisms by polyamide membranes.

Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

Diabetic nephropathy: where hemodynamics meets metabolism.

Diabetic nephropathy (DN), the most common cause of end stage renal disease in developed nations, is thought to result from interactions between metabolic and haemodynamic factors. Specific metabolically driven, glucose dependent pathways are activated within diabetic renal tissues. These pathways induce oxidative stress, polyol pathway flux, hexosamine flux and accumulation of advanced glycated end-products (AGEs). Haemodynamic factors are also implicated in the pathogenesis of DN and include elevations of systemic and intraglomerular pressure and activation of various vasoactive hormone pathways including the renin-angiotensin aldosterone system (RAAS), endothelin and urotensin. These altered hemodynamics act independently and in concert with metabolic pathways, to activate intracellular second messengers such as protein kinase C (PKC) and MAP kinase (MAPK), nuclear transcription factors such as nuclear factor-kappaB (NF-kappaB) and various growth factors such as the prosclerotic cytokines, transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF) and the angiogenic, permeability enhancing growth factor, vascular endothelial growth factor, VEGF. Ultimately these molecular mechanisms lead to increased renal albumin permeability, and extracellular matrix accumulation, which results in increasing proteinuria, glomerulosclerosis and tubulointerstitial fibrosis. In the past, the treatment of diabetic nephropathy has focused on control of hyperglycemia and the interruption of the RAAS with certain anti-hypertensive agents. Newer novel targets, some of which are linked to glucose dependent pathways, appear to be a major focus of new therapies directed against the development and progression of renal damage as a result of diabetes. It is likely that resolution of diabetic nephropathy will require synergistic therapies to target multiple mediators of this disease.

Adult-onset acute tubulointerstitial nephritis and uveitis with Fanconi syndrome. Case report and review of the literature.

We report a case of tubulointerstitial nephritis and uveitis (TINU syndrome) with full type Fanconi syndrome. A 32-year-old woman presented with fatigue, anorexia and weight loss. Laboratory findings showed anemia, polyclonal hypergammaglobulinemia and moderate renal dysfunction. Tubular function abnormalities were normoglycemic glucosuria, panaminoaciduria, phosphaturia and kaliuresis leading to hypokalemia. Renal tubular acidosis and hypouricemia were also evident. Serum antistreptolysin O titer was high. Ocular symptoms (bilateral anterior uveitis) emerged soon after admission. Renal biopsy showed diffuse tubulointerstitial infiltration by lymphocytes and plasma cells without granuloma. Treatment with systemic steroids was given and renal function, and ocular symptom returned to normal with 3 months. Although tubular abnormalities involving TINU syndrome has already been reported, the disease associated with full type Fanconi syndrome has rarely been seen, and systemic steroid may be beneficial in reducing the development of tubulointerstitial injury.

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Cellular distribution of polyphosphoinositides in rat hepatocytes.

The distribution of total phospholipids, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was studied in isolated rat hepatocytes: (i) by mass assay and isotopic labelling in the fractions of plasma membranes, microsomes, mitochondria and nuclei prepared from isolated hepatocytes and (ii) by immunolocalization of PIP2 with a specific antibody (kt3g) in whole hepatocytes and isolated nuclei. Mass measurement and isotopic labelling showed that PIP was distributed in all four fractions. PIP2 was present in the plasma membrane and the nuclei. In whole cells, PIP2 was also detected in the plasma membrane by immunolocalization with the anti-PIP2 antibody kt3g. In unpolarized single hepatocytes, PIP2 distributed evenly throughout the plasma membrane. However, in polarized cell couplets, PIP2 was the most often undetectable in the lateral domain between the cells, and distributed preferentially in the sinusoidal domain of the plasma membrane. These results suggest that hepatocytes segregate PIP2 in particular domains of their plasma membrane. In purified fractions of nuclei, immunolocalization experiments showed that PIP2 was present uniquely in the nuclear envelope.

Simultaneous measurements of exocytosis and intracellular calcium concentration with fluorescent indicators in single pituitary gonadotropes.

Previously, we established a method for the estimation of exocytosis in single gonadotropes using an impermeable fluorescent membrane probe, TMA-DPH. In this study, we have developed a method for the simultaneous measurement of exocytosis and intracellular free Ca2+ concentration ([Ca2+]i) by double-labeling with TMA-DPH and the intracellular Ca2+ probe, Fura-2/AM, using a fluorescence microscope with a 3-wavelength excitation and 2-wavelength emission system. We, therefore, clarified the relationship between spontaneous [Ca2+]i oscillation or gonadotropin releasing hormone (GnRH)-induced intracellular Ca2+ mobilization and exocytosis in gonadotropes. Under resting conditions, some gonadotropes showed various types of spontaneous [Ca2+]i oscillations, while others did not, but all showed basal exocytosis. Each [Ca2+]i peak oscillation did not cause Ca(2+)-regulated exocytosis, and even complete blockage of the [Ca2+]i increase by the intracellular Ca2+ chelator BAPTA/AM had no effect on basal exocytosis. Both GnRH-induced intracellular Ca2+ mobilization and regulated exocytosis showed a similar pattern of peaks and plateaus. Blockage of the [Ca2+]i increase by BAPTA/AM almost completely inhibited the GnRH-stimulated exocytosis. These results show that spontaneous [Ca2+]i oscillations under resting conditions are not linked to regulated or basal exocytosis, and that intracellular Ca2+ mobilization is essential for GnRH-stimulated exocytosis.

Neurotensin-containing projections from the retrosplenial cortex to the anterior ventral thalamic nucleus in the rat.

The neurotensin-containing projections from the retrosplenial cortex to the anterior ventral thalamus were demonstrated by electrolytic lesion studies and fluorescent retrograde tracing combined with immunocytochemistry. Three-to-five-day-old rats were used, because the immunoreactivity of neurotensin fibers in anterior ventral thalamus was the highest at this age. When neurotensin-containing neurons located in layer VI of the retrosplenial cortex were unilaterally destroyed by applying an electrolytic current to the retrosplenial area, the neurotensin fibers in the ipsilateral anterior ventral thalamus decreased dramatically. Unilateral injection of a fluorescent retrograde tracer, Fast Blue, into the anterior ventral thalamus, labeled neurons in the ipsilateral retrosplenial cortex, and many of these cells also had neurotensin-like immunoreactivity. These results suggested that a major origin of the neurotensin fibers in the anterior ventral thalamus was in the ipsilateral retrosplenial granular cortex.

Scanning electron microscope assessment of exocytotic changes in purified gonadotropes.

These studies were undertaken to characterize the exocytotic changes in purified gonadotropes by three-dimensional imaging using scanning electron microscopy. Rat gonadotropes were purified using a fluorescence-activated cell sorter and an argon laser treatment system. The purified gonadotropes were stimulated with GnRH under various conditions and fixed for scanning electron microscopy. After the GnRH stimulation, many 'hole' structures (diameter 0.1-0.5 micron) were observed on the cell surface, and notably the population of cells with 10 or more holes was clearly increased. The pattern of the time-course of the changes in this population was perfectly consistent with the LH secretory profile of pituitary cells, and their formation of the cells with 10 or more holes was completely inhibited by pretreatment with a GnRH antagonist. Our data suggest that the hole structure represents an exocytotic opening site and that regulated exocytosis in purified gonadotropes can be evaluated by scanning electron microscopy. This method may be widely applicable to other endocrine cells.

Characterization of the association of phospholipase C-delta with Alzheimer neurofibrillary tangles.

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that an antibody to the PLC isozyme, PLC-delta, intensely stained neurofibrillary tangles (NFT) in the brain tissue of AD patients [Am. J. Pathol., 139 (1991) 737-742]. To clarify the crucial involvement of abnormal PLC-delta accumulation contributing to the formation of NFT, we performed light and electron microscopic immunocytochemistry. To determine PLC-delta's association with NFT, its resistance to solubilization was also studied. Anti-PLC-delta antibody marked the same NFT-bearing neurons containing tau immunoreactivity with tau more clearly on NFT filaments and PLC-delta covering it superficially at the light microscope level. The double stained preparations with anti-PLC-delta antibody and bFGF binding suggested that PLC-delta is an intracellular marker and is not retained after neuronal death. Employing immunoperoxidase and immunogold electron microscopic immunocytochemistry, we found that the antibody to PLC-delta reacts mostly with amorphous granular materials, and occasionally with some abnormal filaments within NFT. Nevertheless, PLC-delta in NFT was resistant to removal by high salt or ionic detergent, indicating it is an integral NFT component. These results indicate that antigenic determinants unique to PLC-delta are mainly present intraneuronally on the amorphous granular components of NFT as well as the abnormal filaments, suggesting PLC-delta's interactions and possible role in the formation of intraneuronal filamentous inclusions in AD.