Exploring strain-level diversity in the gut microbiome through mucin particle adhesion.

Mucin glycoproteins are a significant source of carbon for the gut bacteria. Various gut microbial species possess diverse hydrolytic enzymes and catabolic pathways for breaking down mucin glycans, resulting in competition for the limited nutrients within the gut environment. Adherence to mucin glycans represents a crucial strategy used by gut microbes to access nutrient reservoirs. Understanding these properties is pivotal for comprehending the survival mechanisms of bacteria in the gastrointestinal tract. However, characterization of individual strains within the vast array of coexisting bacteria in the microbiome is challenging. To investigate this, we developed mucin-immobilized particles by immobilizing porcine gastric mucin (PGM) onto glass beads chemically modified with boronic acid. These PGM-immobilized particles were then anaerobically cultured with human fecal microbiota, and the bacteria adhering to PGM were isolated. Interestingly, the microbiome composition remained largely unchanged irrespective of PGM immobilization. Nonetheless, bacteria isolated from PGM-immobilized glass particles exhibited notably higher N-acetylgalactosaminidase activity compared to the control beads. Furthermore, Bacteroides strains isolated from PGM-immobilized glass particles displayed enhanced adhesive and metabolic properties to PGM. These findings underscore the utility of PGM particles in enriching and isolating specific microbes. Moreover, they highlight substantial differences in microbial properties at the strain level. We anticipate that PGM-immobilized particles will advance culture-based microbiome research, emphasizing the significance of strain-level characterization. IMPORTANCE: Metabolism of mucin glycans by gut bacteria represents a crucial strategy for accessing nutrient reservoirs. The efficacy of mucin glycan utilization among gut bacteria hinges on the metabolic capabilities of individual strains, necessitating meticulous strain-level characterization. In this investigation, we used glass beads chemically immobilized with mucins to selectively enrich bacteria from fecal fermentation cultures, based on their superior adhesion to and metabolism of mucin glycoproteins. These findings lend support to the hypothesis that the physical interactions between bacteria and mucin glycoprotein components directly correlate with their capacity to utilize mucins as nutrient sources. Furthermore, our study implies that physical proximity may significantly influence bacterial nutrient acquisition within the ecosystem, facilitating gut bacteria's access to carbohydrate components.

Immunohistochemical localization of HCA1 receptor in placenta in presence of fetal growth restriction.

INTRODUCTION: Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Fetuses with intrauterine growth restriction are considered to become progressively lactacidemic as well as hypoxic. Roles of lactate in the placenta in the presence of fetal growth restriction (FGR) remain to be clarified. METHODS: Immunohistochemical localization of lactate-related substances, such as a receptor for lactate (hydroxy-carboxylic acid 1 receptor (HCA1 receptor/GPR81)), monocarboxylate transporters (MCTs) for lactate, lactate dehydrogenases (LDHs), and proteins expressed in syncytiotrophoblasts or cytotrophoblasts was examined in placentas of appropriate weight for gestational age (AGA) fetus and those showing FGR. RESULTS: Immunoreactivity for the HCA1 receptor was present in the cytoplasm of some trophoblasts, predominantly localized to their basal (fetus-facing) side, and was frequently colocalized with that for E-cadherin or serine peptidase inhibitor, Kunitz type 1 (SPINT1), a marker protein of cytotrophoblasts. Immunoreactivity for MCT1 and MCT4 was present on the basal and the microvillous (maternal-facing) membranes of trophoblasts in both groups, respectively. Clear immunoreactivity for LDHA and LDHB was also observed in the cytoplasm of trophoblasts, mainly localized to their basal side. However, there were no significant differences in immunohistochemically stained areas of lactate-related substances between AGA and late-onset FGR groups. On the other hand, there were correlations between coefficients of the presence of chorioamnionitis and the values of LDHB and E-cadherin. DISCUSSION: Immunohistochemical localization of the HCA1 receptor was predominantly observed in the cytoplasm located on the basal side of trophoblasts, suggesting a role of lactate in human placental development, including syncytialization.

Transporters, Ion Channels, and Junctional Proteins in Choroid Plexus Epithelial Cells.

The choroid plexus (CP) plays significant roles in secreting cerebrospinal fluid (CSF) and forming circadian rhythms. A monolayer of epithelial cells with tight and adherens junctions of CP forms the blood-CSF barrier to control the movement of substances between the blood and ventricles, as microvessels in the stroma of CP have fenestrations in endothelial cells. CP epithelial cells are equipped with several kinds of transporters and ion channels to transport nutrient substances and secrete CSF. In addition, junctional components also contribute to CSF production as well as blood-CSF barrier formation. However, it remains unclear how junctional components as well as transporters and ion channels contribute to the pathogenesis of neurodegenerative disorders. In this manuscript, recent findings regarding the distribution and significance of transporters, ion channels, and junctional proteins in CP epithelial cells are introduced, and how changes in expression of their epithelial proteins contribute to the pathophysiology of brain disorders are reviewed.

Preferential sugar utilization by bifidobacterial species.

Aim: Bifidobacteria benefit host health and homeostasis by breaking down diet- and host-derived carbohydrates to produce organic acids in the intestine. However, the sugar utilization preference of bifidobacterial species is poorly understood. Thus, this study aimed to investigate the sugar utilization preference (i.e., glucose or lactose) of various bifidobacterial species. Methods: Strains belonging to 40 bifidobacterial species/subspecies were cultured on a modified MRS medium supplemented with glucose and/or lactose, and their preferential sugar utilization was assessed using high-performance thin-layer chromatography. Comparative genomic analysis was conducted with a focus on genes involved in lactose and glucose uptake and genes encoding for carbohydrate-active enzymes. Results: Strains that preferentially utilized glucose or lactose were identified. Almost all the lactose-preferring strains harbored the lactose symporter lacS gene. However, the comparative genomic analysis could not explain all their differences in sugar utilization preference. Analysis based on isolate source revealed that all 10 strains isolated from humans preferentially utilized lactose, whereas all four strains isolated from insects preferentially utilized glucose. In addition, bifidobacterial species isolated from hosts whose milk contained higher lactose amounts preferentially utilized lactose. Lactose was also detected in the feces of human infants, suggesting that lactose serves as a carbon source not only for infants but also for gut microbes in vivo. Conclusion: The different sugar preference phenotypes of Bifidobacterium species may be ascribed to the residential environment affected by the dietary habits of their host. This study is the first to systematically evaluate the sugar uptake preference of various bifidobacterial species.

E-Cadherin Is Expressed in Epithelial Cells of the Choroid Plexus in Human and Mouse Brains.

Evidence showing the functional significance of the choroid plexus is accumulating. Epithelial cells with tight and adherens junctions of the choroid plexus play important roles in cerebrospinal fluid production and circadian rhythm formation. Although specific types of cadherin expressed in adherens junctions of choroid plexus epithelium (CPE) have been examined, they remained uncertain. Recent mass spectrometry and immunolocalization analysis revealed that non-epithelial cadherins, P- and N-cadherins, are expressed in the lateral membrane of CPE, whereas E-cadherin expression has not been confirmed in CPE of humans or mice. In this study, we examined E-cadherin expression in CPE of mice and humans by RT-PCR, immunohistochemical-, and Western blotting analyses. We confirmed, by using RT-PCR analysis, the mRNA expression of E-cadherin in the choroid plexus of mice. The immunohistochemical expression of E-cadherin was noted in the lateral membrane of CPE of mice and humans. We further confirmed, in Western blotting, the specific immunoreactivity for E-cadherin. Immunohistochemically, the expression of E- and N-cadherins or vimentin was unevenly distributed in some CPE, whereas that of E- and P-cadherins or ß-catenin frequently co-existed in other CPE. These findings indicate that E-cadherin is expressed in the lateral membrane of CPE, possibly correlated with the expression of other cadherins and cytoplasmic proteins.

Distribution of Monocarboxylate Transporters in Brain and Choroid Plexus Epithelium.

The choroid plexus (CP) plays central roles in regulating the microenvironment of the central nervous system by secreting the majority of cerebrospinal fluid (CSF) and controlling its composition. A monolayer of epithelial cells of CP plays a significant role in forming the blood-CSF barrier to restrict the movement of substances between the blood and ventricles. CP epithelial cells are equipped with transporters for glucose and lactate that are used as energy sources. There are many review papers on glucose transporters in CP epithelial cells. On the other hand, distribution of monocarboxylate transporters (MCTs) in CP epithelial cells has received less attention compared with glucose transporters. Some MCTs are known to transport lactate, pyruvate, and ketone bodies, whereas others transport thyroid hormones. Since CP epithelial cells have significant carrier functions as well as the barrier function, a decline in the expression and function of these transporters leads to a poor supply of thyroid hormones as well as lactate and can contribute to the process of age-associated brain impairment and pathophysiology of neurodegenerative diseases. In this review paper, recent findings regarding the distribution and significance of MCTs in the brain, especially in CP epithelial cells, are summarized.

Synbiotics of Bifidobacterium breve MCC1274 and lactulose enhances production of tryptophan metabolites in fermented human fecal communities.

Probiotics and prebiotics have beneficial effects on host physiology via metabolites from the gut microbiota in addition to their own. Here, we used a pH-controlled single-batch fermenter as a human gut microbiota model. We conducted fecal fermentation with Bifidobacterium breve MCC1274 (probiotic), lactulose (prebiotic), or a combination of both (synbiotic) to evaluate their influence on the gut environment. Fecal inoculum without the probiotic and prebiotic was used as the control. Principal coordinate analysis (PCoA), based on the composition of gut microbiota, showed a significant difference among the groups. The relative abundance of Bifidobacterium was significantly higher in the synbiotic group, compared to that in the other three treatment groups. The relative abundance of Blautia was the highest in the control group among the four groups. CE-TOFMS and LC-TOFMS showed that the number of metabolites detected in the synbiotic group was the highest (352 in total); 29 of the 310 hydrophilic metabolites and 17 of the 107 lipophilic metabolites were significantly different among the four groups in the Kruskal-Wallis test. A clustering based on 46 metabolites indicated that tryptophan-metabolites such as indole-3-lactic acid (ILA), indole-3-ethanol, and indole-3-carboxaldehyde, were included in a sub cluster composed of metabolites enriched in the synbiotic group. Spermidine, a major polyamine, was enriched in the two groups supplemented with the probiotic whereas spermine was enriched only in the synbiotic group. Not all metabolites enriched in the probiotic and/or synbiotic groups were found in the monocultures of the probiotic strain with or without the prebiotics. This implies that some of the metabolites were produced through the interaction of the fecal microbiota with the inoculated probiotic strain. Co-abundance networking analysis indicated the differences in the correlations between the relative abundance of the fecal microbiota genus and the tryptophan metabolites in each group. There was a strong correlation between ldh4 gene abundance and ILA concentration in the fecal fermentation. The copy number of ldh4 gene was significantly higher in the groups with the probiotic than that in the control group. In conclusion, synbiotics could enhance the production of signaling molecules in the gut environment. Our results provide an insight into more effective administration of probiotics at the molecular level.

Metabolites and Biomarker Compounds of Neurodegenerative Diseases in Cerebrospinal Fluid.

Despite recent advances in diagnostic procedures for neurological disorders, it is still difficult to definitively diagnose some neurodegenerative diseases without neuropathological examination of autopsied brain tissue. As pathological processes in the brain are frequently reflected in the components of cerebrospinal fluid (CSF), CSF samples are sometimes useful for diagnosis. After CSF is secreted from the choroid plexus epithelial cells in the ventricles, some flows in the brain, some is mixed with intracerebral interstitial fluid, and some is excreted through two major drainage pathways, i.e., the intravascular periarterial drainage pathway and the glymphatic system. Accordingly, substances produced by metabolic and pathological processes in the brain may be detectable in CSF. Many papers have reported changes in the concentration of substances in the CSF of patients with metabolic and neurological disorders, some of which can be useful biomarkers of the disorders. In this paper, we show the significance of glucose- and neurotransmitter-related CSF metabolites, considering their transporters in the choroid plexus; summarize the reported candidates of CSF biomarkers for neurodegenerative diseases, including amyloid-ß, tau, α-synuclein, microRNAs, and mitochondrial DNA; and evaluate their potential as efficient diagnostic tools.

Immunohistochemical expression of osteopontin and collagens in choroid plexus of human brains.

Evidence showing the functional significance of the choroid plexus is accumulating. Although it is clinically well-known that calcification is frequently seen in the choroid plexus of aged human brains, it is unclear why calcification occurs in the aged choroid plexus and what exert effects on the calcification has. In this study, immunohistochemical localizations of collagens and other molecules related to fibrosis or calcification were investigated on the choroid plexus of autopsied human brains. Densely fibrous or calcified materials were located in the stroma just below the epithelial cells of the choroid plexus of all human brains examined. Immunoreactivity for collagen type I was identified in the stroma just below the epithelial cells, consistent with the densely fibrous or calcified area, whereas that for collagen type III was observed in almost all stroma other than the densely fibrous or calcified areas. Linear or membranous immunoreactivity for collagen type IV was intermittently localized on the epithelium-facing side of the materials, suggesting an injured basement membrane. In addition, clear immunoreactivity for osteopontin was localized on the epithelium-facing side of the fibrous or calcified materials as well as in the cytoplasm of epithelial cells. These findings indicate that collagen type I exists in contact with osteopontin in and around the densely fibrous or calcified materials in the choroid plexus. They suggest that the densely fibrous or calcified materials are deposited in the subepithelial stroma just below an injured basement membrane of epithelial cells via the collagen type I and osteopontin.

Computational Prediction of Compound-Protein Interactions for Orphan Targets Using CGBVS.

A variety of Artificial Intelligence (AI)-based (Machine Learning) techniques have been developed with regard to in silico prediction of Compound-Protein interactions (CPI)-one of which is a technique we refer to as chemical genomics-based virtual screening (CGBVS). Prediction calculations done via pairwise kernel-based support vector machine (SVM) is the main feature of CGBVS which gives high prediction accuracy, with simple implementation and easy handling. We studied whether the CGBVS technique can identify ligands for targets without ligand information (orphan targets) using data from G protein-coupled receptor (GPCR) families. As the validation method, we tested whether the ligand prediction was correct for a virtual orphan GPCR in which all ligand information for one selected target was omitted from the training data. We have specifically expressed the results of this study as applicability index and developed a method to determine whether CGBVS can be used to predict GPCR ligands. Validation results showed that the prediction accuracy of each GPCR differed greatly, but models using Multiple Sequence Alignment (MSA) as the protein descriptor performed well in terms of overall prediction accuracy. We also discovered that the effect of the type compound descriptors on the prediction accuracy was less significant than that of the type of protein descriptors used. Furthermore, we found that the accuracy of the ligand prediction depends on the amount of ligand information with regard to GPCRs related to the target. Additionally, the prediction accuracy tends to be high if a large amount of ligand information for related proteins is used in the training.

Discovery of Novel eEF2K Inhibitors Using HTS Fingerprint Generated from Predicted Profiling of Compound-Protein Interactions.

Background: Eukaryotic elongation factor 2 kinase (eEF2K) regulates the elongation stage of protein synthesis by phosphorylating eEF2, a process related to various diseases including cancer and cardiovascular and neurodegenerative diseases. In this study, we describe the identification of novel eEF2K inhibitors using high-throughput screening fingerprints (HTSFP) generated from predicted profiling of compound-protein interactions (CPIs). Methods: We utilized computationally generated HTSFPs referred to as chemical genomics-based fingerprint (CGBFP). Generally, HTSFPs are generated from multiple biochemical or cell-based assay data. On the other hand, CGBFPs are generated from computational prediction of CPIs using the Chemical Genomics-Based Virtual Screening (CGBVS) method. Therefore, CGBFPs do not have missing information mainly caused by the absence of assay data. Results: Chemogenomics-Based Similarity Profiling (CGBSP) of the screening library (2.6 million compounds) yielded 27 compounds which were evaluated for in vitro eEF2K inhibitory activity. Three compounds with interesting results were identified. Compounds 2 (IC50 = 11.05 µM) and 4 (IC50 = 43.54 µM) are thieno[2,3-b]pyridine derivatives that have the same scaffolds with a known eEF2K inhibitor, while compound 13 (IC50 = 70.13 µM) was a new thiophene-2-amine-type eEF2K inhibitor. Conclusions: CGBSP supplied an efficient strategy in the identification of novel eEF2K inhibitors and provided useful scaffolds for optimization.

Growth-promoting effect of alginate on Faecalibacterium prausnitzii through cross-feeding with Bacteroides.

Faecalibacterium prausnitzii is a commensal gut bacterium that is thought to provide protection against inflammatory diseases. However, this bacterium is extremely oxygen sensitive, which limits its industrial application as a probiotic. The use of prebiotics to increase the abundance of this bacterium in the gut is an alternative strategy to achieve its possible health-promoting effect. We evaluated nine substances as candidate prebiotics for F. prausnitzii using a pH-controlled single-batch fermenter as a human gut microbiota model. Of them, alginate markedly increased the relative abundance of F. prausnitzii, as determined by the significant increase in the number of 16S rRNA sequences corresponding to this bacterial taxon in the fecal fermentation samples detected by real-time PCR. However, F. prausnitzii strains were incapable of utilizing alginate in monoculture, implying that an interaction with another gut microbe was required. There was a positive correlation between the relative abundance of F. prausnitzii and that of Bacteroides when cultured in medium containing alginate as the sole carbon source, indicative of cross-feeding between these bacteria. Interestingly, the ratio of acetic acid, a known substrate for F. prausnitzii, produced by Bacteroides was significantly higher in the alginate-containing medium than in media containing other prebiotic candidates. Bacterially degraded alginate oligosaccharides (AOS) remained in the medium after Bacteroides monoculture, and an isolate of F. prausnitzii was able to utilize a portion of them. Genomic sequencing revealed that the strain that consumed the AOS contained an ATP-binding cassette transporter, an alginate lyase, and AlgQ1/2 homologs encoding solute-binding proteins. Furthermore, in real-time PCR analyses, AlgQ1/2 homologs were detected in fecal samples collected from 309 of 452 (68.4%) Japanese subjects. Thus, the products of alginate assimilation by Bacteroides may promote the growth of F. prausnitzii.

Immunoreactivity of receptor and transporters for lactate located in astrocytes and epithelial cells of choroid plexus of human brain.

Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Cerebral ischemia or hypoxia is believed to become progressively lactacidemic. Monocarboxylate transporters (MCTs) in endothelial cells are essential for the transport of lactate from the blood into the brain. In addition, it is considered that MCTs located in astrocytic and neuronal cells play a key role in the shuttling of energy metabolites between neurons and astrocytes. However, roles of lactate in the brain remain to be clarified. In this study, the localization of lactate transporters and a receptor for cellular uptake of lactate was immunohistochemically examined in autopsied human brains. Immunoreactivity for MCT1 was observed in the apical cytoplasmic membrane of some epithelial cells in the choroid plexus as well as astrocytes and the capillary wall, whereas that for MCT4 was found in the basolateral cytoplasmic membrane of small number of epithelial cells as well as astrocytes and the capillary wall. In addition, immunoreactivity for the hydroxy-carboxylic acid 1 receptor (HCA1 receptor), a receptor for cellular uptake of lactate, was also found on the basolateral cytoplasmic membrane of epithelial cells as well as astrocytic and neuronal cells. Immunoreactivity for lactate dehydrogenase (LDH)-B was observed in the cytoplasm of epithelial cells in the choroid plexus as well as astrocytes and the capillary wall. These immunohistochemical findings indicate the localization of MCT1, MCT4, the HCA1 receptor, and LDH-B in epithelial cells of the choroid plexus as well as astrocytes, and suggest the transport of intravascular lactate into the brain through epithelial cells of the choroid plexus as well as cerebral vessels and the possibility of lactate being utilized in epithelial cells.

Glucose, Fructose, and Urate Transporters in the Choroid Plexus Epithelium.

The choroid plexus plays a central role in the regulation of the microenvironment of the central nervous system by secreting the majority of the cerebrospinal fluid and controlling its composition, despite that it only represents approximately 1% of the total brain weight. In addition to a variety of transporter and channel proteins for solutes and water, the choroid plexus epithelial cells are equipped with glucose, fructose, and urate transporters that are used as energy sources or antioxidative neuroprotective substrates. This review focuses on the recent advances in the understanding of the transporters of the SLC2A and SLC5A families (GLUT1, SGLT2, GLUT5, GLUT8, and GLUT9), as well as on the urate-transporting URAT1 and BCRP/ABCG2, which are expressed in choroid plexus epithelial cells. The glucose, fructose, and urate transporters repertoire in the choroid plexus epithelium share similar features with the renal proximal tubular epithelium, although some of these transporters exhibit inversely polarized submembrane localization. Since choroid plexus epithelial cells have high energy demands for proper functioning, a decline in the expression and function of these transporters can contribute to the process of age-associated brain impairment and pathophysiology of neurodegenerative diseases.

Sodium/glucose cotransporter 2 is expressed in choroid plexus epithelial cells and ependymal cells in human and mouse brains.

Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease-modifying effects of anti-diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti-diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription-polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains.

Iron plays essential roles in the central nervous system. However, how the iron level is regulated in brain cells including glia and neurons remains to be fully clarified. In this study, the localizations of hepcidin, ferroportin, and hephaestin, which are known to be involved in iron efflux, were immunohistochemically examined in autopsied human brains. Immunoreactivities for hepcidin and ferroportin were observed in granular structures within the cytoplasm of reactive astrocytes and epithelial cells of the choroid plexus. Granular structures showing immunoreactivities for hepcidin and ferroportin were also stained with antibodies for early endosome antigen 1 (EEA1). In addition, immunoreactivity for hephaestin was observed in the cytoplasm of epithelial cells of the choroid plexus as well as reactive astrocytes. Immunoreactivity for hephaestin in the cytoplasm of reactive astrocytes was occasionally colocalized with immunoreactivity for EEA1, while that of hephaestin was frequently observed in the cytoplasm showing no immunoreactivity for EEA1. These findings suggest that immunoreactivities for hepcidin and ferroportin are localized in close proximity to granular structures showing immunoreactivity for EEA1 in the cytoplasm of human brain astrocytes. They also suggest that immunoreactivity of hephaestin is localized in the cytoplasm of the choroid plexus epithelium as well as reactive astrocytes of human brains.

Disturbance of Intracerebral Fluid Clearance and Blood-Brain Barrier in Vascular Cognitive Impairment.

The entry of blood-borne macromolecular substances into the brain parenchyma from cerebral vessels is blocked by the blood-brain barrier (BBB) function. Accordingly, increased permeability of the vessels induced by insult noted in patients suffering from vascular dementia likely contributes to the cognitive impairment. On the other hand, blood-borne substances can enter extracellular spaces of the brain via endothelial cells at specific sites without the BBB, and can move to brain parenchyma, such as the hippocampus and periventricular areas, adjacent to specific sites, indicating the contribution of increased permeability of vessels in the specific sites to brain function. It is necessary to consider influx and efflux of interstitial fluid (ISF) and cerebrospinal fluid (CSF) in considering effects of brain transfer of intravascular substances on brain function. Two pathways of ISF and CSF are recently being established. One is the intramural peri-arterial drainage (IPAD) pathway of ISF. The other is the glymphatic system of CSF. Dysfunction of the two pathways could also contribute to brain dysfunction. We review the effects of several kinds of insult on vascular permeability and the failure of fluid clearance on the brain function.

Histomorphometry of ectopic mineralization using undecalcified frozen bone sections.

To investigate the correlation between mineral formation and enhanced expressions of some proteins using undecalcified frozen bone sections. Histological studies have revealed that some proteins, such as BMP2, BMPR1A, and Connexin 43, are expressed in and around sites of ectopic ossification. However, the relationship between the expressed proteins considered to be associated with the ossification and mineral formation in vivo is not clear. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1)-mutant spinal hyperostotic TWY mice and ICR mice as controls were euthanized after calcein labeling, and undecalcified frozen sections were obtained from the middle thoracic spine. Intervertebral disc areas were examined histologically and by measuring calcein-labeled areas and areas showing immunoreactivity for BMP2, BMPR1A, and Connexin 43. Calcein-labeled areas, indicating mineralization in the ectopic mineralization sites, were significantly larger in the mutant mice than in controls. The expression of Connexin 43 was elevated in the annulus fibrosus. Increases in the calcein-labeled areas was not correlated with increases in the areas showing immunoreactivity for Connexin 43 in the annulus fibrosus. There was no statistical correlation between enhanced immunohistochemical expression and elevated calcein-labeled areas. By applying the morphometrical analysis method using undecalcified frozen sections to ENPP1-mutant mice, quantitative evaluation of the mineralization and proteins expressed in the surrounding area in the same animal became possible.

Immunoreactivity of urate transporters, GLUT9 and URAT1, is located in epithelial cells of the choroid plexus of human brains.

It has been suggested that urate plays a protective role in neurons, while hyperuricemia is correlated with atherosclerosis and cardiovascular disease. However, whether there is a system that directly transports urate into the brain remains to be clarified. In this study, the localization of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), which are known to be representative reabsorptive urate transporters, was immunohistochemically examined in autopsied human brains. Immunoreactivity of GLUT9 was observed on the apical side of the cytoplasm of epithelial cells in the choroid plexus and in the cilia of ependymal cells of the human brain. Immunoreactivity of URAT1 was observed on the basolateral side of the cytoplasm of epithelial cells in the choroid plexus. In addition, immunoreactivity of GLUT9 and URAT1 was not observed in microvessels of the human brains. The choroid plexus and renal proximal tubule were similar in having a polarized distribution of these two transporters with the two transporters on opposite membranes, but the two transporters' distribution differs between the choroid plexus and the kidney in terms of which membrane (apical/basal) expresses which transporter. These findings support the hypothesis of the direct transport of intravascular urate into the central nervous system through the choroid plexus.

Glucose transporter 8 immunoreactivity in astrocytic and microglial cells in subependymal areas of human brains.

Glucose transporter 8 (GLUT8), a glucose/fructose transporter, has been shown to be expressed in neuronal cells in several brain areas. A recent immunohistochemical study has shown the presence of GLUT8 in the cytoplasm of epithelial cells of the choroid plexus and ependymal cells. In this study, localization of GLUT8 in glial cells was investigated using immunohistochemical methods. Immunoreactivity for GLUT8 was observed in cells showing astrocytic or microglial structural features located around the lateral ventricles. Confocal microscopic examination revealed that subependymal GLUT8-positive cells with large amounts of cytoplasm mainly show clear immunoreactivity for vimentin, while they were also colocalized with weak immunoreactivity for glial fibrillary acidic protein (GFAP) within the cytoplasm of some cells. In addition, some GLUT8-positive cells with small amounts of cytoplasm and small nuclei showed CD68 or HLA-DR immunoreactivity, indicating them to be cells of microglia/macrophage lineage. These findings suggest that glucose/fructose is transported into the cytoplasm of vimentin- or GFAP-positive astrocytic and CD68- or HLA-DR-positive microglial cells located around the lateral ventricle.