Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma.

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.

Involvement of membrane palmitoylated protein 2 (MPP2) in the synaptic molecular complex at the mouse cerebellar glomerulus.

We previously reported that the membrane skeletal protein 4.1G in the peripheral nervous system transports membrane palmitoylated protein 6 (MPP6), which interacts with the synaptic scaffolding protein Lin7 and cell adhesion molecule 4 (CADM4) in Schwann cells that form myelin. In the present study, we investigated the localization of and proteins related to MPP2, a highly homologous family protein of MPP6, in the cerebellum of the mouse central nervous system, in which neurons are well organized. Immunostaining for MPP2 was observed at cerebellar glomeruli (CG) in the granular layer after postnatal day 14. Using the high-resolution Airyscan mode of a confocal laser-scanning microscope, MPP2 was detected as a dot pattern and colocalized with CADM1 and Lin7, recognized as small ring/line patterns, as well as with calcium/calmodulin-dependent serine protein kinase (CASK), NMDA glutamate receptor 1 (GluN1), and M-cadherin, recognized as dot patterns, indicating the localization of MPP2 in the excitatory postsynaptic region and adherens junctions of granule cells. An immunoprecipitation analysis revealed that MPP2 formed a molecular complex with CADM1, CASK, M-cadherin, and Lin7. Furthermore, the Lin7 staining pattern showed small rings surrounding mossy fibers in wild-type CG, while it changed to the dot/spot pattern inside small rings detected with CADM1 staining in MPP2-deficient CG. These results indicate that MPP2 influences the distribution of Lin7 to synaptic cell membranes at postsynaptic regions in granule cells at CG, at which electric signals enter the cerebellum.

Three-dimensional structural analysis of mitochondria composing each subtype of fast-twitch muscle fibers in chicken.

In a previous study, the three-dimensional structures of mitochondria in type I and type IIb muscle fibers of chicken were analyzed. The study reported differences in the shape of the mitochondria and the distribution of lipid droplets. In this study, we three-dimensionally analyzed mitochondria and lipid droplets of type II muscle fiber subtypes IIa, IIb, and IIc of chicken lateral iliotibial muscle in the same field of view using correlative light electron microscopy (CLEM) and array tomography methods. The reconstructed images showed that the mitochondria of type IIa muscle fiber were thick and aligned along the myofibrils, and many lipid droplets were embedded in the mitochondria. The mitochondria of type IIb muscle fibers were intermittent, aligned along the myofibrils, and showed contact between adjacent horizontal mitochondria. No lipid droplets were observed in type IIb muscle fiber. In type IIc muscle fiber, we observed irregularly shaped mitochondria with small diameters aligned along the myofibrils. Lipid droplets not only were embedded in the mitochondria but also existed independently in some cases. The combination of array tomography and CLEM methods enabled three-dimensional electron microscopic observation of mitochondria in different subtypes of type II muscle fibers. The subtypes of type II muscle fibers differed in mitochondrial occupancy and morphology and in lipid droplet distribution, and characteristics that had been demonstrated biochemically were also demonstrated ultrastructurally.

The unique physiological features of the broiler pectoralis major muscle as suggested by the three-dimensional ultrastructural study of mitochondria in type IIb muscle fibers.

Typical skeletal muscles are composed of mixed muscle fiber types, which are classified as slow-twitch (type I) and fast-twitch (type II) fibers, whereas pectoralis major muscles (PMs) in broiler chickens are 100% composed of type IIb fast-twitch fibers. Since metabolic properties differ among muscle fiber types, the combination of muscle fiber types is involved in physiological functions and pathological conditions in skeletal muscles. In this study, using serial block-face scanning electron microscopy, we compared three-dimensional (3D) mitochondrial properties in type IIb fibers in broiler PMs and those in type I fibers of broiler gastrocnemius muscles (GMs) heterogeneously composed of slow- and fast-twitch muscle fibers. In type I fibers in the GMs, elongated mitochondria with numerous interconnections to form a substantial network among myofibrils were observed. Along with lipid droplets sandwiched by mitochondria, these features are an adaptation to effective oxidative respiration and constant oxidative damage in slow-twitch muscle fibers. In contrast, type IIb fibers in the PMs showed small and ellipsoid-shaped mitochondria with few interconnections and no lipid droplets, forming a sparse network. The mitochondrial spatial network comprises of active mitochondrial dynamics to reduce mitochondrial damage; therefore, type IIb fibers possess physiologically low capacity to maintain mitochondrial wellness due to static mitochondrial dynamics. Based on 3D mitochondrial properties, we discussed the contrasting physiological functions between type I and IIb fibers and proposed a high contractile power and low stress resistance as unique physiological properties of broiler PMs.

Non-microtubule tubulin-based backbone and subordinate components of postsynaptic density lattices.

A purification protocol was developed to identify and analyze the component proteins of a postsynaptic density (PSD) lattice, a core structure of the PSD of excitatory synapses in the central nervous system. "Enriched"- and "lean"-type PSD lattices were purified by synaptic plasma membrane treatment to identify the protein components by comprehensive shotgun mass spectrometry and group them into minimum essential cytoskeleton (MEC) and non-MEC components. Tubulin was found to be a major component of the MEC, with non-microtubule tubulin widely distributed on the purified PSD lattice. The presence of tubulin in and around PSDs was verified by post-embedding immunogold labeling EM of cerebral cortex. Non-MEC proteins included various typical scaffold/adaptor PSD proteins and other class PSD proteins. Thus, this study provides a new PSD lattice model consisting of non-microtubule tubulin-based backbone and various non-MEC proteins. Our findings suggest that tubulin is a key component constructing the backbone and that the associated components are essential for the versatile functions of the PSD.

Three-dimensional ultrastructure reconstruction of tendinous components at the bifurcation of the bovine superficial digital flexor tendon using array and STEM tomographies.

Tendons transmit force from muscle to bone for joint movement. Tenocytes are a specialized type of fibroblast that produces collagen fibrils in tendons. Their cytoplasmic processes form a network surrounding collagen fibrils to define a collagen fibre. Glycosaminoglycan (GAG) chains link collagen fibrils and adhere at the D-band of the collagen fibril. In this study, we used array and scanning transmission electron microscope (STEM) tomographies to reconstruct the three-dimensional ultrastructure of tenocytes, collagen fibres, collagen fibrils and GAG chains at the bifurcation of the bovine hindlimb superficial digital flexor tendon (SDFT). Collagen fibrils comprising a collagen fibre were not aligned uniformly and had at least two running directions. Spindle-shaped tenocytes were arranged along the long axis of a plurality of collagen fibres, where two groups of collagen fibrils with oblique directions to each other exhibited an oblique overlap of the two collagen fibril layers. Collagen fibrils with different running directions were observed in separating layers of about 300 nm in thickness and had diameters of 0-200 nm. About 40% of all collagen fibrils had a peak in the range of 20-40 nm. STEM analysis of the same site where the crossing of collagen fibres was observed by transmission electron microscopy demonstrated the outline of collagen fibrils with a clear D-banding pattern at a regular interval. Collagen fibrils were reconstructed three-dimensionally using continuous images acquired by STEM tomography, which confirmed that the collagen fibrils at the crossing sites did not orientate in layers, but were woven one by one. Higher magnification observation of GAG chains attached between the crossing collagen fibrils revealed numerous GAG chains arranged either vertically or obliquely on collagen fibrils. Furthermore, GAG chains at the cross of collagen fibrils connected the closest D-bands. GAG chains are thought to be universally present between collagen fibrils of the tendon. These observations by array and STEM tomographies increase our knowledge of the anatomy in the bifurcation of the bovine hindlimb SDFT and demonstrate the utility of these new imaging technologies.

Ultrastructural study of the three-dimensional tenocyte network in newly hatched chick Achilles tendons using serial block face-scanning electron microscopy.

The lateral cytoplasmic processes of tenocytes extend to form three-dimensional network surrounding collagen fibers. It is unknown whether connections between two cytoplasmic processes involve overlapping of the processes or merely surface contact. In this study, the two-dimensional and three-dimensional structure of tenocytes in the Achilles tendons of the newly hatched chicks were studied using transmission electron microscopy and serial block face-scanning electron microscopy. Observation of the two-dimensional structures revealed various forms of cellular connections, including connections between the cytoplasmic processes of adjacent tenocytes and between the cytoplasmic process of tenocytes and fibroblasts. Three-dimensional observation showed spike-like cytoplasmic processes extending from one tenocyte that interlocked with cytoplasmic processes from other tenocytes. Cytoplasmic processes from each tenocyte within the chick tendons interlocked to ensure a tight cell-to-cell connection around growing collagen fibers. A cellular network formed by these cytoplasmic processes surrounds each collagen fiber. Cell-cell junctions, which were suggested to be gap junctions, observed at sites of cytoplasmic process overlap most likely represent the major route for communication between tenocytes associated with fibroblasts, enabling vital signals important for maintaining the cell and tendon integrity to be transmitted.

Distribution of lanthanum carbonate in the gastric mucosa confirmed by electron microscopy with a magnified endoscopy: a case report and literature review.

We describe the case of a 70-year-old man with diabetic nephropathy undergoing hemodialysis. Four years following hemodialysis, he started taking lanthanum carbonate 1500 mg/day and lansoprazole 30 mg/day. Nine years following hemodialysis, he underwent screening esophagogastroduodenoscopy, which demonstrated the presence of the whitish cobblestone-like mucosa in the gastric corpus and multiple reddish depressed lesions with annular whitish mucosa in the antrum. With magnified narrow-band imaging endoscopy, a yellowish-white substance was observed in the villous structure, and subepithelial vessels were observed on the yellowish-white substance. Biopsies were taken from the whitish cobblestone-like mucosa of the upper corpus, a reddish depressed part of the antrum. Histologically, aggregates of cells containing amphophilic fine granular material were found in the mucosal interstitium. These cells stained positive for CD68 and were identified as histiocytes. Since he had been taking lanthanum carbonate for 5 years, we considered the possibility of histiocyte-mediated phagocytosis of lanthanum. Digital mapping via scanning electron microscopy with energy-dispersive X-ray spectrometry showed the presence of lanthanum and phosphorus in the interstitium and cytoplasm of histiocytes. The white, rough mucosa in the gastric body appeared 6 months following the commencement of lanthanum administration and still exists 3 years and 5 months after discontinuation of lanthanum.

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high-pressure freezing unit.

The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.

Protein components of post-synaptic density lattice, a backbone structure for type I excitatory synapses.

It is essential to study the molecular architecture of post-synaptic density (PSD) to understand the molecular mechanism underlying the dynamic nature of PSD, one of the bases of synaptic plasticity. A well-known model for the architecture of PSD of type I excitatory synapses basically comprises of several scaffolding proteins (scaffold protein model). On the contrary, 'PSD lattice' observed through electron microscopy has been considered a basic backbone of type I PSDs. However, major constituents of the PSD lattice and the relationship between the PSD lattice and the scaffold protein model, remain unknown. We purified a PSD lattice fraction from the synaptic plasma membrane of rat forebrain. Protein components of the PSD lattice were examined through immuno-gold negative staining electron microscopy. The results indicated that tubulin, actin, α-internexin, and Ca2+ /calmodulin-dependent kinase II are major constituents of the PSD lattice, whereas scaffold proteins such as PSD-95, SAP102, GKAP, Shank1, and Homer, were rather minor components. A similar structure was also purified from the synaptic plasma membrane of forebrains from 7-day-old rats. On the basis of this study, we propose a 'PSD lattice-based dynamic nanocolumn' model for PSD molecular architecture, in which the scaffold protein model and the PSD lattice model are combined and an idea of dynamic nanocolumn PSD subdomain is also included. In the model, cytoskeletal proteins, in particular, tubulin, actin, and α-internexin, may play major roles in the construction of the PSD backbone and provide linker sites for various PSD scaffold protein complexes/subdomains.

Using Light and Electron Microscopy to Estimate Structural Variation in Thylakoid Membranes.

The shapes of chloroplasts and the architectures of internal thylakoid membranes are altered by growth and environmental changes ( Lichtenthaler et al., 1981 ; Kutik, 1985; Terashima and Hikosaka, 1995). These morphological alterations proceed via transitional intermediates, during which dynamic and heterogeneous thylakoid membranes are observed in cells ( Nozue et al., 2017 ). Light microscopy is useful for the detection of morphological differences in chloroplasts. The thylakoid architecture of such morphologically variable chloroplasts is confirmed by transmission electron microscopy (TEM). The method of monitoring structural variation by light microscopy in combination with electron microscopy is described.

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy.

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.

Membrane-associated activation of cholesterol α-glucosyltransferase, an enzyme responsible for biosynthesis of cholesteryl-α-D-glucopyranoside in Helicobacter pylori critical for its survival.

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-D-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme's localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

A thin carbon-fiber web as a scaffold for bone-tissue regeneration.

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high-purity carbon fibers in a nanoscale structure. When the thin carbon-fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP-2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone-tissue compatibility. Further, the rhBMP-2/TCFW composite repairs a critical-size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.

Human amniotic mesenchymal cells differentiate into chondrocytes.

Recently, cartilage diseases have been treated by auto- or allogenic chondrocyte transplantation. However, such treatments are limited by the necessity of having a large amount of cells for transplantation, the risk of rejection, and donor shortage. Since the human amnion is immune-privileged tissue suitable for allotransplantation, the potential of human amniotic mesenchymal cells (HAMc) to differentiate into chondrocytes was assessed. The expression of gene encoding transcription factors SOXs and bone morphogenetic proteins (BMPs) as well as BMP receptors were assessed. Chondrocyte phenotype was characterized by positive expression of the cartilage marker genes collagen type II and aggrecan by RT-PCR, collagen type II protein were analyzed by immunofluorescence analysis. HAMc expressed chondrocyte-related genes, including SOXs, BMPs, as well as BMP receptors. Collagen type II and aggrecan were detected after the induction of chondrogenesis with BMP-2. HAMc, transplanted into noncartilage tissue of mice with BMP-2, or implanted with collagen-scaffold into the defects generated in a rat's bone, underwent morphological changes with deposition of collagen type II. These results showed that HAMc have the potential to differentiate into chondrocytes in vitro and in vivo, suggesting that they have therapeutic potential for the treatment of damaged or diseased cartilage.

Quantitative elemental analysis on aluminum accumulation by HVTEM-EDX in liver tissues of mice orally administered with aluminum chloride.

Quantitative elemental analysis on Al was carried out by high-accelerating voltage transmission electron microscopy (HVTEM) equipped with energy-dispersive X-ray microanalysis (EDX) using an accelerating voltage at 300 kV with high permeability in 1-mum-thick samples obtained from mice administered with aluminum chloride solution for 3, 9, and 17 weeks. By light microscopic observation, no morphological changes were observed in the hepatocytes and macrophages in the liver tissues of mice that were administered with excess Al as compared with the normal control mice. In contrast, by electron microscopic observation, ultrastructural changes were observed in the lysosomes in the hepatocytes as well as the pinocytotic vesicles in the macrophages in the experimental animals. Therefore, the concentrations of Al detected in lysosomes in hepatocytes and pinocytotic vesicles in macrophages of livers of mice administered with Al were measured in relationship to those administration periods. Moreover, transitional changes of hepatocyte lysosome ratios by image analysis and the macrophage counts in the unit area increased in liver tissues of mice administered with Al as compared with normal control mice. From the results, it was demonstrated that hepatocyte lysosome ratio and macrophage count increased in liver tissues of treated mice during those short-term excessive Al administration periods. It was also clarified that the concentrations of Al in both hepatocytes and macrophages increased as observed by HVTEM-EDX. In conclusion, Al accumulated in hepatocytes and macrophages at 3 and 9 weeks administration, while the ultrastructural changes remained in the hepatocytes and macrophages. In contrast, Al concentration did not increase in the liver at 17 weeks administration.

Property analysis of ectopic calcification in the carpal tunnel identification of apatite crystals: a case report.

INTRODUCTION: A 64-year-old woman presented with symptoms of subacute exacerbation of a year-long carpal tunnel syndrome that was caused by a large calcified mass in the tunnel. CONCLUSION: The resected mass consisted of very tiny rods, and x-ray diffraction analysis, as well as the component analysis using energy dispersive x-ray microanalysis, revealed the mass to be most compatible with apatite. The back-scattered electron images suggested that precipitation might be a mechanism for development of the calcified mass.

Localization of translational components at the ultramicroscopic level at postsynaptic sites of the rat brain.

We investigated the localization of components of translational machinery and their regulators in the postsynaptic region. We examined several components, especially those involved in translational regulation: components of (1) MAPK-Mnk-eIF4E, (2) PI3-kinase-PDK-Akt/PKB-FRAP/mTOR-PHAS/4EBP, (3) p70S6K-S6 ribosomal protein and (4) eEF2 kinase/CaMKIII-eEF2 pathways. Western blotting detected all the components examined in the synaptic fractions, and their differential localization to the synaptic subcompartments: initiation or elongation factors, except for eIF5, were detected predominantly in the dendritic lipid raft fraction, which contained ER marker proteins. In contrast, most of their regulatory kinases were distributed to both the postsynaptic density (PSD) and the dendritic lipid raft fractions, or enriched in the former fraction. Localization of eIF4E at synaptic sites was further examined immunohistochemically at the electron microscopic level. The eIF-4E-immunoreactivity was localized to the postsynaptic sites, especially to the microvesicle-like structures underneath the postsynaptic membrane in the spine, some of which were localized in close proximity to PSD. These results suggest that the postsynaptic local translational system, in at least four major regulatory pathways, is similar to those in the perinuclear one, and that it takes place, at least partly, immediately beneath the postsynaptic membrane. The results also suggest the presence of ER-associated type of translational machinery at the postsynaptic sites.

Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats.

Local injection of mu-opioid receptor specific neurotoxin, dermorphin-saporin, into the striatum resulted in selective degeneration of striatal neurons in the patch compartment. We analyzed subsequent anterograde degeneration of axons and terminals at light and electron microscopic level. Light microscopic examination after silver impregnation method revealed that degenerating axons and terminals arising from the striatal patch compartment were distributed in the globus pallidus, entopeduncular nucleus, and substantia nigra. They were found in both pars reticulata and compacta of the substantia nigra. Electron microscopic examination revealed that the degenerating axon terminals contained large pleomorphic vesicles and formed symmetric synapses on dendrites. The present results suggest that patch neurons expressing mu-opioid receptor send projection fibers to multiple nuclei of the basal ganglia.

Detection of aluminium by energy dispersive X-ray microanalysis at high accelerating voltages with semi-thin sections of biological sample.

Aluminium (Al) was detected in semi-thin sections of three organs, the duodenum, liver and kidney, of ddY strain mouse by energy dispersive X-ray microanalysis at high accelerating voltages around 300 kV. Firstly, to determine the conditions best for detecting Al, several adult ddY mice were injected intraperitoneally with aluminium chloride (AlCl3) and the duodenums were fixed, embedded and sectioned at various thicknesses and subjected to energy dispersive X-ray microanalysis at various accelerating voltages from 100 to 400 kV. From the results obtained, 1.0 microm-thick sections observed at 300 kV resulted in the highest peak-counts/background ratios and were shown to be the most suitable for X-ray microanalysis. Secondly, ddY mice aged four weeks were administered orally with 2% AlCl3 at pH 2.5 for two weeks and the three organs (duodenum, liver and kidney) were subjected to X-ray microanalysis under the same condition found above. The results were compared with light microscopic Al staining of the same tissues. Aluminium was detected in lysosomes of the three kinds of tissues with higher sensitivity by energy dispersive X-ray microanalysis by light microscopic observation. From the results, it was suggested that Al dissolved in acidic water was absorbed in the duodenum and accumulated in the liver and kidney.