O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

Total transferrin in cerebrospinal fluid is a novel biomarker for spontaneous intracranial hypotension.

Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Patients with SIH experience postural headaches, nausea, etc., due to CSF hypovolemia. Imaging studies and clinical examinations, such as radioisotope (RI) scintigraphy, are useful for diagnosing SIH. However, 20-30% of patients do not show typical morphology and clinical test results. We previously reported that CSF contains transferrin (Tf) isoforms:"brain-type" Tf derived from the choroid plexus and "serum-type" Tf derived from blood. We showed that both isoforms increased in the CSF of patients with SIH by Western blotting. In the present study, we demonstrate that conventional ELISA for quantifying total Tf is useful for diagnosing SIH more accurately than Western blotting. In addition, SIH with chronic subdural hematoma (CSDH) was also accurately diagnosed.　Total Tf in the CSF can serve as a useful biomarker for diagnosing SIH with or without CSDH.

A study of the relationship between nuclear contour thickening, nuclear enlargement and human papillomavirus infection in squamous cells.

OBJECTIVE: To investigate the relationship between the morphological features of nuclear enlarged cells and human papillomavirus (HPV) infection in negative for intraepithelial lesion or malignancy (NILM) and atypical squamous cells of undetermined significance (ASC-US). METHODS: In total, 128 Papanicolaou specimens comprising 41 ASC-US cases and 87 NILM cases were examined. Cell morphological analysis was performed using both area ratio (nuclear area in cells with nuclear enlargement/nuclear area in normal intermediate cells) and nuclear contour thickening. High-risk HPV was detected using the Uniplex E6/E7 polymerase chain reaction assay and logistic regression analyses of factors related to high-risk HPV infection were performed. RESULTS: Nuclear contour thickening was present in 57.7% (64/111 cells) of high-risk HPV positive cases and 21.8% (69/317 cells) of high-risk HPV negative cases. There was a statistically significant association (P = 0.01) between high-risk HPV infection and nuclear contour thickening. Nuclear contour thickening was approximately one-third higher in NILM cases than in ASC-US cases (odds ratio, 0.371; 95% confidence interval, 0.208-0.662) and three times higher in high-risk HPV-positive cases than in high-risk HPV-negative cases (odds ratio, 2.831; 95% confidence interval, 1.591-5.039). CONCLUSION: Our results suggest that nuclear contour thickening in nuclear enlarged cells in NILM and ASC-US cases may be a cellular finding associated with HPV infection.

A Comparison of Cytomorphological Features of ASC-H Cells Based on Histopathological Results Obtained from a Colposcopic Target Biopsy Immediately after Pap smear Sampling.

Background: To compare the cytomorphological features of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) observed in a liquid-based Pap smear with the histopathological features observed in a concurrent colposcopic biopsy specimen obtained immediately after obtaining the Pap smear. Methods: Cytomorphological features such as cytoplasmic differentiation, nuclear/cytoplasm (N/C) ratio, chromatin pattern, thickening of nuclear contour, and the appearance of the nucleolus of 247 ASC-H obtained from 25 liquid-based Pap smear ASC-H cases were compared with those of the cells obtained from biopsied samples. Human papillomavirus (HPV) infection was tested for 39 HPV genotypes using Uniplex E6/E7 polymerase chain reaction method. Results: Of the 25 ASC-H cases, 22 (88%) showed cervical intraepithelial neoplasia grade 1 or greater (CIN1+) and 3 (12%) were benign. HPV infection was detected in 100% CIN1+ cases and 66.7% benign cases. Significant differences such as marked hyperchromasia, thickened nuclear contour, and prominent nucleoli were observed between ASC-H cases with CIN1+ and the benign cases. Conclusion: The presence of small dysplastic cells displaying marked hyperchromasia, thickening of nuclear contour, and prominent nucleoli on Pap smear strongly suggest the presence of CIN in ASC-H cases.

Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling.

Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-ß (IFN-ß) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-ß production, impaired IFN-ß production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.

Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination.

DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here, we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR, as evidenced by the data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and to preventing early steps in HR by deleting XRCC3 suppressed the nonviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.

Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells.

BACKGROUND: We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cells after immunization with T-cell-dependent antigen. Zizimin2 was revealed to be a new family member of Dock (dedicator of cytokinesis), Dock11, which is the guanine nucleotide exchange factor for Cdc42, a low-molecular-weight GTPase. However, the molecular function of Zizimin2 in acquired immunity has not been elucidated. RESULTS: In this study, we show that the protein expression of Zizimin2, which is also restricted to lymphoid tissues and lymphocytes, is reduced in aged mice. Over-expression of full-length Zizimin2 induced filopodial formation in 293T cells, whereas expression of CZH2 domain inhibited it. Stimulation of Fcγ receptor and Toll-like receptor 4 triggered Zizimin2 up-regulation and Cdc42 activation in bone marrow-derived dendritic cells. CONCLUSIONS: These data suggest that Zizimin2 is an immune-related and age-regulated guanine nucleotide exchange factor, which facilitates filopodial formation through activation of Cdc42, which results in activation of cell migration.

Inflammatory alterations of the extracellular matrix in the tumor microenvironment.

Complex interactions between cancer cells and host stromal cells result in the formation of the "tumor microenvironment", where inflammatory alterations involve the infiltration of tumor-associated fibroblasts and inflammatory leukocytes that contribute to the acquisition of malignant characteristics, such as increased cancer cell proliferation, invasiveness, metastasis, angiogenesis, and avoidance of adaptive immunity. The microenvironment of a solid tumor is comprised not only of cellular compartments, but also of bioactive substances, including cytokines, growth factors, and extracellular matrix (ECM). ECM can act as a scaffold for cell migration, a reservoir for cytokines and growth factors, and a signal through receptor binding. During inflammation, ECM components and their degraded fragments act directly and indirectly as inflammatory stimuli in certain cases and regulate the functions of inflammatory and immune cells. One such ECM component, hyaluronan, has recently been implicated to modulate innate immune cell function through pattern recognition toll-like receptors and accelerate the recruitment and activation of tumor-associated macrophages in inflamed cancers. Here, we will summarize the molecular mechanism linking inflammation with ECM remodeling in the tumor microenvironment, with a particular emphasis on the role of hyaluronan in controlling the inflammatory response.

RAP80 acts independently of BRCA1 in repair of topoisomerase II poison-induced DNA damage.

The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80(-/-) cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80(-/-)BRCA1(-/-) cells were more sensitive to etoposide than were BRCA1(-/-) cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80(-/-) cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1.

Immunohistochemical and morphologic basis for glutamate signaling in the rat stomach.

Physiologic studies conducted in rats have demonstrated that afferent fibers of the gastric branch of the vagus nerve increase their firing rate with the intragastric administration of the amino acid glutamate, and the increased firing rate is blocked by the depletion of serotonin (5-HT), administration of the blocker for the serotonin type-3 receptor (SR3), or nitric oxide synthase (NOS). To understand glutamate signaling in the gastric mucosa at the cellular level, we have been studying rats as an animal model using anatomic and immunohistochemical procedures. Our results have indicated that 5-HT-immunoreactive (ir) cells are present in the superficial part of the gastric mucosal epithelium and in the base of the fundic glands, whereas immunoreactivity for SR3 is localized in the neck and its vicinity of the fundic glands. Further, NOS1/neuronal NOS-ir cells with a bipolar shape are located in the lamina propria where a dense network of neuronal cells is present. These results suggest that complex cellular events take place during intragastric glutamate signaling.

A translocation causing increased alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism.

Phosphate homeostasis is central to diverse physiologic processes including energy homeostasis, formation of lipid bilayers, and bone formation. Reduced phosphate levels due to excessive renal loss cause hypophosphatemic rickets, a disease characterized by prominent bone defects; conversely, hyperphosphatemia, a major complication of renal failure, is accompanied by parathyroid hyperplasia, hyperparathyroidism, and osteodystrophy. Here, we define a syndrome featuring both hypophosphatemic rickets and hyperparathyroidism due to parathyroid hyperplasia as well as other skeletal abnormalities. We show that this disease is due to a de novo translocation with a breakpoint adjacent to alpha-Klotho, which encodes a beta-glucuronidase, and is implicated in aging and regulation of FGF signaling. Plasma alpha-Klotho levels and beta-glucuronidase activity are markedly increased in the affected patient; unexpectedly, the circulating FGF23 level is also markedly elevated. These findings suggest that the elevated alpha-Klotho level mimics aspects of the normal response to hyperphosphatemia and implicate alpha-Klotho in the selective regulation of phosphate levels and in the regulation of parathyroid mass and function; they also have implications for the pathogenesis and treatment of renal osteodystrophy in patients with kidney failure.

alpha-Klotho as a regulator of calcium homeostasis.

alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an alpha-Kl- and Na+,K+-ATPase-dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of alpha-Kl in the regulation of calcium metabolism.

Cell-cell interaction-dependent regulation of N-acetylglucosaminyltransferase III and the bisected N-glycans in GE11 epithelial cells. Involvement of E-cadherin-mediated cell adhesion.

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and alpha1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

Usefulness of Auto Cyto Fix (membrane filter method) for the application of immunohistochemistry.

We developed the Auto Cyto Fix (ACF) 1000 as an experimental model, which was adapted as a membrane filter method for automatically making smear preparations fixed with 95% ethanol. In this study, immunohistochemistry was applied to the cells smeared on the membrane filter, and effectiveness was examined. Twenty-four effusions and HeLa 229 cells were used. These cell samples were produced by the ACF1000 on the membrane filter and stained by the Papanicolaou method. After observation, these were decolored and stained by immunohistochemical techniques. Antibodies included CEA, EMA, and MIB-1. CEA and EMA were detected by the indirect method, and were colored by DAB, AEC, and new fuchsin. MIB-1 was detected by the immunoperoxidase (LSAB) and the immunofluorescence method. A uniformly stabilized immunoreaction was obtained and was equivalent to or better than that of the conventional technique. In addition, there was no background staining on the membrane filter. Based on these results, the membrane filter preparation produced by the ACF1000 was also effectively applied to immunohistochemical observations.