Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration.

Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound healing. Thrombomodulin (TM) is an integral membrane protein that participates in cell-cell adhesion through its extracellular lectin-like domain. However, the molecular basis of TM-mediated cell-cell adhesion is poorly understood. Here, we demonstrate that TM is linked to the actin cytoskeleton via ezrin. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified (522)RKK(524) as important ezrin-binding residues. In epidermal epithelial A431 cells, TM colocalized with ezrin and actin filaments at cell-cell contacts. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, stimulated the interaction between ezrin and TM. In skin wound healing of mice, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient melanoma A2058 cells promoted collective cell migration. In summary, TM, which associates with ezrin and actin filaments, maintains epithelial morphology and promotes collective cell migration.

The enhancement of biofilm formation in Group B streptococcal isolates at vaginal pH.

Group B streptococcus (GBS) is a common asymptomatic colonizer in acidic vagina of pregnant women and can transmit to newborns, causing neonatal pneumonia and meningitis. Biofilm formation is often associated with bacterial colonization and pathogenesis. Little is known about GBS biofilm and the effect of environmental stimuli on their growth along with biofilm formation. The objective of this study was to investigate the survival and biofilm formation of GBS, isolated from pregnant women, in nutrient-limited medium under various pH conditions. Growth and survival experiments were determined by optical density and viable counts. Crystal violet staining, scanning electron microscopy, and atomic force microscopy (AFM) were used to analyze the capacity of biofilm production. Our results showed that GBS isolates proliferated with increasing pH with highest maximum specific growth rate (µmax) at pH 6.5, but survived at pH 4.5 for longer than 48 h. Biofilm formation of the 80 GBS isolates at pH 4.5 was significantly higher than at pH 7.0. This difference was confirmed by two other methods. The low elastic modulus obtained from samples at pH 4.5 by AFM revealed the softness of biofilm; in contrast, little or no biofilm was measured at pH 7.0. Under acidic pH, the capability of biofilm formation of serotypes III and V showed statistically significant difference from serotypes Ia and Ib. Our finding suggested that survival and enhanced biofilm formation at vaginal pH are potentially advantageous for GBS in colonizing vagina and increase the risk of vaginosis and neonatal infection.

SEPT12 mutations cause male infertility with defective sperm annulus.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

Experimental phage therapy in treating Klebsiella pneumoniae-mediated liver abscesses and bacteremia in mice.

Intragastric inoculation of mice with Klebsiella pneumoniae can cause liver abscesses, necrosis of liver tissues, and bacteremia. A newly isolated phage (φNK5) with lytic activity for K. pneumoniae was used to treat K. pneumoniae infection in an intragastric model. Both intraperitoneal and intragastric administration of a single dose of φNK5 lower than 2 × 10(8) PFU at 30 min after K. pneumoniae infection was able to protect mice from death in a dose-dependent manner, but the efficacy achieved with a low dose of φNK5 by intragastric treatment provided the more significant protection. Phage φNK5 administered as late as 24 h after K. pneumoniae inoculation was still protective, while intraperitoneal treatment with phage was more efficient than intragastric treatment as a result of the dissemination of bacteria into the circulation at 24 h postinfection. Surveys of bacterial counts for mice treated with φNK5 by the intraperitoneal route revealed that the bacteria were eliminated effectively from both blood and liver tissue. K. pneumoniae-induced liver injury, such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase and inflammatory cytokine production, was significantly inhibited by φNK5 treatment. These data suggest that a low dose of φNK5 is a potential therapeutic agent for K. pneumoniae-induced liver infection.

The expression level of septin12 is critical for spermiogenesis.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Low substratum rigidity of collagen gel promotes ERK phosphorylation via lipid raft to augment cell migration.

Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MbetaCD (Methyl-beta-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbetaCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.

Biocompatible bacteria@Au composites for application in the photothermal destruction of cancer cells.

We have successfully transformed the infectious E. coli bacteria into biocompatible bacteria@Au composites for photothermal therapy.

Enhanced myometrial autophagy in postpartum uterine involution.

OBJECTIVE: To understand the mechanisms of postpartum uterine involution, we investigated the uterine myometrial changes during pregnancy and the postpartum period. MATERIALS AND METHODS: Nine groups of uterine myometrial samples from mice (n = 4) were collected on gestational Day 0 (nonpregnant), Day 1, Day 2, Day 7, Day 14, and Day 21 and on postpartum Day 1, Day 2, and Day 7. Human samples of uterine myometrium on term (n = 1) and postpartum Day 1 (n = 2) were also collected. Ki-67 immunostaining was used to determine myometrial proliferation. For cell hypertrophy analysis, organelle proteins, ß-actin, prohibin, calnexin, and golgin-97 were analyzed by Western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and evaluation of activated caspase-3 expression by Western blot analysis assay were used to detect apoptosis. Autophagy was assayed via the evaluation of LC3 expression by Western blotting, immunohistochemistry, and autophagosomes by electron microscopy. RESULTS: Uterine myocytes proliferated during the early stage of gestation with a peak at Day 2, whereas myocyte hypertrophy with increased cellular organelle production occurred gradually in later stages of pregnancy. Postpartum autophagy developed abruptly in uterine myocytes without obvious apoptosis. CONCLUSION: Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.

Riboflavin-ultraviolet-A-induced collagen cross-linking treatments in improving dentin bonding.

OBJECTIVES: To evaluate the collagen cross-linkers, riboflavin-ultraviolet-A (RF/UVA) and glutaraldehyde, with regard to their efficacy in cross-linking the dentinal collagen and improving dentin bonding. METHODS: Glutaraldehyde and different RF/UVA protocols (0.1%RF/1-minUV, 0.1%RF/2-minUV, and 1%RF/1-minUV) were first evaluated by gel electrophoresis to determine their abilities of collagen cross-linking. The mechanical properties of acid-etched dentin receiving these cross-linking treatments were examined in either dry or wet condition by a nanoindentation test. Fifteen teeth with exposed occlusal dentin received the microtensile bond strength (µTBS) test. The teeth were primed either with RF/UVA or glutaraldehyde, followed by adhesive treatment and composite restorations, and then cut into resin-dentin microbeams. Half of the microbeams received the µTBS test after 24h, and the other half received test after 5000 thermocycles. Nanoleakage at the bond interface was examined under TEM. The alignments of collagen fibrils in the hybrid layers were also defined by an image analysis. RESULTS: Gel electrophoresis showed that glutaraldehyde induced strong collagen gelation, while RF/UVA generated milder collagen cross-linking. Glutaraldehyde, 0.1%RF/2-min-UVA, and 1%RF/1-minUV showed higher stiffness compared to untreated and 0.1%RF/1-minUV in wet condition. All the crosslinking treatments improved early µTBS, but 0.1%RF/2-minUVA treatment maintained high µTBS after theromocycles. Under TEM, glutaraldehyde-treated dentin showed dense and enclosed collagen network on the adhesive interface. 0.1%RF/2-minUVA showed the least nanoleakage, and this could be associated with the suspended collagen fibrils in the hybrid layer. SIGNIFICANCE: 0.1%RF/2-minUVA treatment enhanced resin-dentin bond possibly through enhancing the stiffness and maintaining the expanding collagen matrix in the hybrid layer.

Targeting cathepsin S induces tumor cell autophagy via the EGFR-ERK signaling pathway.

Cathepsin S is a cellular cysteine protease, which is frequently over-expressed in human cancer cells and plays important role in tumor metastasis. However, the role of cathepsin S in regulating cancer cell survival and death remains undefined. The aim of this study was to determine whether targeting cathepsin S could induce autophagy/apoptosis in cancer cells. In this study, we demonstrated that targeting cathepsin S by either specific small molecular inhibitors or cathepsin S siRNA induced autophagy and subsequent apoptosis in human cancer cells, and the induction of autophagy was dependent on the phosphorylation of EGFR and activation of the EGFR-related ERK/MAPK-signaling pathway. In conclusion, the current study reveals that cathepsin S plays an important role in the regulation of cell autophagy through interference with the EGFR-ERK/MAPK-signaling pathway.

SEPTIN12 genetic variants confer susceptibility to teratozoospermia.

It is estimated that 10-15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12(+/+)/Septin12(+/-) chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage. Ex vivo experiment showed truncated SEPT12 inhibits filament formation in a dose-dependent manner. This study provides the first causal link between SEPTIN12 genetic variant and male infertility with distinctive sperm pathology. Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

Novel phosphoinositide 3-kinase/mTOR dual inhibitor, NVP-BGT226, displays potent growth-inhibitory activity against human head and neck cancer cells in vitro and in vivo.

PURPOSE: Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently accounts for the tumorigenesis in head and neck cancer. To develop a new treatment, we investigated the effect of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in head and neck cancer cells. EXPERIMENTAL DESIGN: The in vitro antitumor effect of BGT226 was determined in various cancer cell lines. Animal models were also applied to examine drug potency. The inhibitory ability of BGT226 on the PI3K/AKT/mTOR signaling pathway was analyzed. RESULTS: The growth inhibition assay revealed that BGT226 was active against all tested cancer cell lines. Cross-resistance was not observed in the cisplatin-resistant cell line. The activation of the AKT/mTOR signal cascade was suppressed by BGT226 in a concentration- and time-dependent manner. Flow cytometric analysis revealed an accumulation of cells in the G(0)-G(1) phase with concomitant loss in the S-phase. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and the analysis of caspase 3/7 and PARP indicated that BGT226 induced cancer cell death through an apoptosis-independent pathway. BGT226 induced autophagy as indicated by the aggregation and upregulation of the microtubule-associated protein light chain 3B-II, and p62 degradation. Gene silencing of Beclin1 or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited the BGT226-induced autophagy and led to the retrieval of colony survival. In a xenografted animal model, BGT226 significantly delayed tumor growth in a dose-dependent manner, along with suppressed cytoplasmic expression of p-p70 S6 kinase and the presence of autophagosome formation. CONCLUSIONS: These data indicate that BGT226 is a potential drug in the treatment of head and neck cancer.

Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors.

Arsenic trioxide (As(2)O(3)) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As(2)O(3) as a promising treatment for APL, several studies investigated the use of As(2)O(3) as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As(2)O(3) with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As(2)O(3) exhibits a synergistic interaction with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As(2)O(3) with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As(2)O(3) and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As(2)O(3) with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As(2)O(3). These findings will be useful in designing future clinical trials of combination chemotherapy with As(2)O(3) and BCNU, with the potential for broad use against a variety of solid tumors.

Association of cord plasma leptin with birth size in term newborns.

BACKGROUND: Leptin is secreted from adipose tissue and plays an important role in obesity. Recent studies have shown that the relationship between Leptin and body fat mass may have ethnic differences. The purpose of our study was to investigate the relationship between venous umbilical cord plasma Leptin and anthropometric markers in term healthy Taiwanese newborns. METHODS: Umbilical venous plasma samples were obtained from 98 term neonates (48 males and 50 females) and leptin Levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: Umbilical cord plasma Levels of leptin were significantly higher in the female neonates than in males (p<0.001). The large-for-gestationaL age and appropriate-for-gestational age newborns had significantly higher Leptin cord plasma levels than the small-for-gestational age newborns (p<0.01 and p<0.05, respectively). In both male and female neonates, umbilical Leptin Levels showed significant positive correlations with birth weight and birth Length. Multiple Linear regression analysis revealed that birth weight was the only significant predictor of umbilical cord plasma Leptin levels in both male and female neonates. However, the slopes of the regressions between Leptin and birth weight in male and female neonates were not different. CONCLUSION: In Taiwanese healthy term neonates, leptin umbilical cord plasma Levels are associated with sex and birth weight of the neonate. The relationship between Leptin and birth weight may differ among different ethnic groups. These findings imply that the relationship between leptin and body fat mass may develop early in life.

Nestin serves as a prosurvival determinant that is linked to the cytoprotective effect of epidermal growth factor in rat vascular smooth muscle cells.

Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H(2)O(2) treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3 and PARP were activated in nestin-depleted VSMCs following H(2)O(2) treatments, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well known that EGF serves as a survival factor in rat VSMCs. Here, we show that the cytoprotective effect of EGF was prevented by nestin RNAi. In addition, the inhibition of Cdk5 prevented Bcl-2 phosphorylation and enhanced H(2)O(2)-induced caspase-3 activation as well as subsequent DNA fragmentation. Taken together, these results provide evidence for another cytoprotective role of EGF in that it is mediated through its stimulation of nestin expression which leads to the prevention of caspase activation by Cdk-5-induced Bcl-2 phosphorylation in rat VSMCs.

Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

Synthesis and characterization of a novel paramagnetic macromolecular complex [Gd(TTDASQ-protamine)].

Adenocarcinomas in rats and humans frequently contain perivascular, degranulating mast cells that release heparin. Protamine is a low-molecular weight, cationic polypeptide that binds to heparin and neutralizes its anticoagulant properties. A novel magnetic resonance imaging (MRI) contrast agent containing protamine was synthesized. TTDASQ, the derivative of TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid), was also synthesized and the kinetic stability of [Gd(TTDASQ)]- chelate containing phosphate buffer and ZnCl2 to measure the relaxation rate (R1) at 20 MHz was studied by transmetallation with Zn(II). The water-exchange rate (k(ex)298) of [Gd(TTDASQ)]- is 6.4 x 10(6) s(-1) at 25.0 +/- 0.1 degrees C which was obtained from the reduced 17O relaxation rates (1/T(1r) and 1/T(2r)) and chemical shift (omega(r)) of H(2)17O, and it is compared with that previously reported for the other gadolinium(III) complex, [Gd(DO3ASQ)]. The binding affinity assay showed that the (TTDASQ)3-pro19 has higher activity toward heparin. On the other hand, the effect of heparin on the relaxivity of the [Gd(TTDASQ)3-pro19] conjugate shows the binding strength (K(A)) is 7669 dm3 mol(-1) at pH 7.4 and the relaxivity (r(b)1) of the [Gd(TTDASQ)3-pro19]-heparin adduct is 30.9 dm3 mmol(-1) s(-1).

Hemoglobin promotes Abeta oligomer formation and localizes in neurons and amyloid deposits.

The objective of this study was to search for brain-specific binding proteins that participated in Abeta aggregation. Immunoprecipitation of Abeta in Alzheimer's brain homogenate revealed a major co-precipitating 16-kDa protein band, which was identified through mass spectrometry as hemoglobin (Hb) alpha and beta chains. Hemoglobin was distributed in Alzheimer's disease (AD) patients in a brain region-dependent manner, with the highest levels in the hippocampus and parietal gray (PG) matter, followed by parietal white matter (PW), and the lowest in cerebellum (Cb). AD parietal gray and white matters exhibited higher Hb levels than those in the nondemented (ND) group. Likewise, RT-PCR revealed that the Hb mRNA levels in AD inferior temporal gyri were higher than those of ND subjects. Furthermore, Hb was shown to promote Abeta oligomer formation. Immunohistochemical studies indicated that Hb was localized within the cytosol of pyramidal neurons in the hippocampus, suggesting a potential source of intracerebral Hb. Finally, double immunofluorescent assay confirmed the co-localization of Hb with senile plaques (SP) and cerebral amyloid angiopathy (CAA). We propose that an elevation in brain Hb via circulation leakage or perturbations of Hb gene regulation may participate in AD pathogenesis.