Abundant extrasynaptic expression of α3ß4-containing nicotinic acetylcholine receptors in the medial habenula-interpeduncular nucleus pathway in mice.

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and ß4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3ß4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and ß4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and ß4 subunits disappeared in α5ß4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that α3ß4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3ß4-containing nAChRs in the crucial pathway of nicotine dependence.

Association between the Angle of the Left Subclavian Artery and Procedural Time for Percutaneous Coronary Intervention in Patients with Acute Coronary Syndrome.

Background: The effect of left subclavian artery tortuosity during percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS) remains unclear. Methods: Of 245 ACS patients (from November 2019 and May 2021), 79 who underwent PCI via a left radial approach (LRA) were included. We measured the angle of the left subclavian artery in the coronal view on CT imaging as an indicator of the tortuosity and investigated the association between that angle and the clinical variables and procedural time. Results: Patients with a left subclavian artery angle of a median of <70 degrees (severe tortuosity) were older (75.4 ± 11.7 vs. 62.9 ± 12.3 years, P < 0.001) and had a higher prevalence of female sex (42.1% vs. 14.6%, P=0.007), hypertension (94.7% vs. 75.6%, P=0.02), and subclavian artery calcification (73.7% vs. 34.2%, P < 0.001) than those with that ≥70 degrees. The left subclavian artery angle correlated negatively with the sheath cannulation to the first balloon time (ρ = -0.51, P < 0.001) and total procedural time (ρ = -0.32, P=0.004). A multiple linear regression analysis revealed that the natural log transformation of the sheath insertion to first balloon time was associated with a subclavian artery angle of <70 degrees (ß = 0.45, P < 0.001). Conclusion: Our study showed that lower left subclavian artery angles as a marker of the tortuosity via the LRA were strongly associated with a longer sheath insertion to balloon time and subsequent entire procedure time during the PCI.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

Protective effect of the Impella on the left ventricular function after acute broad anterior wall ST elevation myocardial infarctions with cardiogenic shock: cardiovascular magnetic resonance imaging strain analysis.

BACKGROUND: The clinical efficacy of the Impella for high-risk percutaneous coronary intervention (PCI) and cardiogenic shock remains under debate. We thus sought to investigate the protective effects on the heart with the Impella's early use pre-PCI using cardiac magnetic resonance imaging (CMRI). METHODS: We retrospectively evaluated the difference in the subacute phase CMR imaging results (19 ± 9 days after admission) between patients undergoing an Impella (n = 7) or not (non-Impella group: n = 18 [12 intra-aortic balloon pumps (1 plus veno-arterial extracorporeal membrane oxygenation) and 6 no mechanical circulation systems]) in broad anterior ST-elevation myocardial infarction (STEMI) cases. A mechanical circulation system was implanted pre-PCI. RESULTS: No differences were found in the door-to-balloon time, peak creatine kinase, and hospital admission days between the Impella and non-Impella groups; however, the CMRI-derived left ventricular ejection fraction was significantly greater (45 ± 13% vs. 34 ± 7.6%, P = 0.034) and end-diastolic and systolic volumes smaller in the Impella group (149 ± 29 vs. 187 ± 41 mL, P = 0.006: 80 ± 29 vs. 121 ± 40 mL, P = 0.012). Although the global longitudinal peak strain did not differ, the global radial (GRS) and circumferential peak strain (GCS) were significantly higher in the IMPELLA than non-IMPELLA group. Greater systolic and diastolic strain rates (SRs) in the Impella than non-Impella group were observed in non-infarcted rather than infarcted areas. CONCLUSIONS: Early implantation of an Impella before PCIs for STEMIs sub-acutely prevented cardiac dysfunction through preserving the GRS, GCS, and systolic and diastolic SRs in the remote myocardium. This study provided mechanistic insight into understanding the usefulness of the Impella to prevent future heart failure.

Prognostic Significance of a Combination of Cardiogenic Shock and the Critical Culprit Lesion Location in ST-Elevation Myocardial Infarctions.

Both cardiogenic shock (CS) and critical culprit lesion locations (CCLLs), defined as the left main trunk and proximal left anterior descending coronary artery, are associated with worse outcomes in ST-elevation myocardial infarctions (STEMIs). We aimed to examine how the combination of CS and/or CCLLs affected the prognosis in Japanese STEMI patients in the primary percutaneous coronary intervention era (PPCI-era). The subjects included 624 STEMI patients admitted to our hospital between January 2013 and April 2020. They were divided into four groups according to the combination of CS and CCLLs: CS (-) CCLL (-) group [n = 405], CS (-) CCLL (+) group [n = 150], CS (+) CCLL (-) group [n = 25], and CS (+) CCLL (+) group [n = 44]. The cumulative incidences of all-cause death at 30 days and 1 year were 3.5% and 6.4% in the CS (-) CCLL (-), 3.3% and 5.6% in the CS (-) CCLL (+), 32.0% and 32.0% in the CS (+) CCLL (-), and 50.0% and 65.9% in the CS (+) CCLL (+) group, respectively. After a multivariate adjustment, the CS (+) CCLL (+) group was independently associated with all-cause death (hazard ratio: 17.00, 95% confidence interval: 7.12-40.59 versus the CS (-) CCLL (-) group). In the CS (+) CCLL (+) group, compared to years 2013-2017, the IMPELLA begun to be used (44.4% versus 0%), and intra-aortic balloon pumps significantly decreased (44.4% versus 92.3%) during years 2018-2020, while the medications upon discharge did not significantly differ. The 30-day mortality was numerically lower during years 2018-2020 than years 2013-2017 (Log-rank test, P = 0.092). In conclusion, the prognosis of STEMIs varies greatly depending on the combination of CS and CCLLs, and in particular, patients with both CS and CCLLs had the poorest prognosis during the modern PPCI-era.

Impact of the COVID-19 outbreak on hospitalizations and outcomes in patients with acute myocardial infarction in a Japanese Single Center.

There are a few Japanese data regarding the incidence and outcomes of acute myocardial infarction (AMI) after the coronavirus disease 2019 (COVID-19) outbreak. We retrospectively reviewed the data of AMI patients admitted to the Nihon University Itabashi Hospital after a COVID-19 outbreak in 2020 (COVID-19 period) and the same period from 2017 to 2019 (control period). The patients' characteristics, time course of admission, diagnosis, and treatment of AMI, and 30-day mortality were compared between the two period-groups for both ST-segment elevation myocardial infarction (STEMI) and non-STEMI (NSTEMI), respectively. The AMI inpatients decreased by 5.7% after the COVID-19 outbreak. There were no differences among most patient backgrounds between the two-period groups. For NSTEMI, the time from the symptom onset to admission was significantly longer, and that from the AMI diagnosis to the catheter examination tended to be longer during the COVID-19 period than the control period, but not for STEMI. The 30-day mortality was significantly higher during the COVID-19 period for NSTEMI (23.1% vs. 1.9%, P = 0.004), but not for STEMI (9.4% vs. 8.3%, P = 0.77). In conclusion, hospitalizations for AMI decreased after the COVID-19 outbreak. Acute cardiac care for STEMI and the associated outcome did not change, but NSTEMI outcome worsened after the COVID-19 outbreak, which may have been associated with delayed medical treatment due to the indirect impact of the COVID-19 pandemic.

Absence of coronary angioscopy-derived in-stent thrombi is associated with major bleeding events in acute myocardial infarction.

BACKGROUND AND AIMS: The optimal duration of dual antiplatelet therapy for acute myocardial infarction is controversial because the bleeding risk outweighs the thromboembolic risk. We hypothesized that an in-stent thrombus (IS-thrombus) detected by coronary angioscopy (CAS) after stent implantation would be associated with high bleeding risk. METHODS: This study included 208 patients who underwent CAS at 2 weeks after stent implantation for an acute myocardial infarction. The study was approved by the ethics committee at the Nihon University Itabashi Hospital (reference number RK-200714-10). RESULTS: In 84 patients, in whom no IS-thrombus was identified in the culprit vessel using CAS, the major bleeding event rate was significantly higher than that in patients with IS-thrombi (n = 124). However, no difference was detected in major adverse cardiovascular events (MACE; stroke, hospitalization for a non-fatal myocardial infarction/unstable angina, target lesion revascularization, and cardiovascular death). After adjustments by the propensity score based on patient characteristics, the absence of IS-thrombi remained an independent predictor of major bleeding events (hazard ratio 4.73, 95% confidence interval 2.04-11.00, p < 0.001). CONCLUSIONS: The absence of CAS-detected IS-thrombi in the subacute phase was independently associated with future major bleeding events, but not with MACE. These findings may help optimize the duration of dual antiplatelet therapy.

Anticoagulant therapy with dual antiplatelet for left ventricular thrombus following acute myocardial infarction.

Two men aged 71 and 62 years were admitted for ST elevation myocardial infarction and percutaneous coronary intervention was performed to the occlusion of left anterior descending artery. Echocardiogram showed an akinetic or a dyskinetic movement of left ventricular anterior wall with mural thrombus on admission in Case 1 and 10 days from admission in Case 2. A direct oral anticoagulant (DOAC) in addition to dual antiplatelet therapy (DAPT) in both patients was started successfully for the resolution of left ventricular thrombus 3 weeks after the initiation of DOAC in Case 1, and 2 weeks after the initiation of DOAC in Case 2. However, the dose of DOAC was decreased and aspirin was stopped in Case 1 with HAS-BLED score five due to colon polyp bleeding, and there was no bleeding complication in Case 2 with HAS-BLED score two during this triple therapy. The duration of triple therapy was 2 months in Case 1 and 6 months in Case 2, and of DOAC therapy was total 6 months in both cases. .

Novel Interleukin-6 Inducible Gene PDZ-Binding Kinase Promotes Tumor Growth of Multiple Myeloma Cells.

[Figure: see text] Multiple myeloma (MM) remains an intractable hematological malignancy, despite recent advances in anti-MM drugs. Here, we show that role of PDZ binding kinase (PBK) in MM tumor growth. We identified that interleukin-6 (IL-6) readily increases PBK expression. Kaplan-Meier analysis showed that the MM patients with higher expression of PBK have a significant shorter survival time compared with those with moderate/lower expression of PBK. Knockout of PBK dramatically suppressed in vivo tumor growth in MM cells, while genome editing of PBK changing from asparagine to serine substitution (rs3779620) slightly suppresses the tumor formation. Mechanistically, loss of PBK increased the number of apoptotic cells with concomitant decrease in the phosphorylation level of Stat3 as well as caspase activities. A novel PBK inhibitor OTS514 significantly decreased KMS-11-derived tumor growth. These findings highlight the novel oncogenic role of PBK in tumor growth of myeloma, and it might be a novel therapeutic target for the treatment of patients with MM.

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

ERO1α is a novel endogenous marker of hypoxia in human cancer cell lines.

BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells.

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-ß1 modification and signalling in HCT116 colorectal cancer cells.

Endoplasmic reticulum disulphide oxidase 1α (ERO1α) is an oxidase localized in the endoplasmic reticulum that plays a role in the formation of disulphide bonds of secreted and cell-surface proteins. We previously showed that ERO1α is overexpressed in various types of cancer and we further identified ERO1α expression as a novel factor related to poor prognosis in cancer. However, the biological functions of ERO1α in cancer remain unclear. Here, we investigated the cell biological roles of ERO1α in the human colon-cancer cell line HCT116. ERO1α knockout (KO) by using CRISPR/Cas9 resulted in decreased tumourigenicity in vivo and reduced cell proliferation only under hypoxia in vitro, which suggested that ERO1α promotes cancer progression specifically in a low-oxygen environment. Thus, we evaluated the function of ERO1α in cell proliferation under hypoxia, and found that under hypoxic conditions, ERO1α KO resulted in a contact-inhibited morphology and diminished motility of cells. We further showed that ERO1α KO induced a change in integrin-ß1 glycosylation and thus an attenuation of cell-surface integrin-ß1 expression, which resulted in the aforementioned phenotype. Our study has established a previously unrecognized link between ERO1α expression and integrin activation, and thus provides new evidence for the effectiveness of ERO1α-targeted therapy for colorectal carcinoma.

Spatiotemporal Regulation of Hsp90-Ligand Complex Leads to Immune Activation.

Although heat shock proteins (HSPs) primarily play a pivotal role in the maintenance of cellular homeostasis while reducing extracellular as well as intracellular stresses, their role in immunologically relevant scenarios, including activation of innate immunity as danger signals, antitumor immunity, and autoimmune diseases, is now gaining much attention. The most prominent feature of HSPs is that they function both in their own and as an HSP-ligand complex. We here show as a unique feature of extracellular HSPs that they target chaperoned molecules into a particular endosomal compartment of dendritic cells, thereby inducing innate and adaptive immune responses via spatiotemporal regulation.

Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid ß-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid ß-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aß generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aß in vivo.

Deficiency of NOX1/nicotinamide adenine dinucleotide phosphate, reduced form oxidase leads to pulmonary vascular remodeling.

OBJECTIVE: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. APPROACH AND RESULTS: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. CONCLUSIONS: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.

Evaluation of annexin A5 as a biomarker for Alzheimer's disease and dementia with lewy bodies.

BACKGROUND: Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Aß42). Using a cell culture model we previously identified annexin A5, a Ca(2+), and phospholipid binding protein, as an AD biomarker. Plasma level of annexin A5 was significantly higher in AD patients compared to that in a control group. On the other hand, AD has been identified to share a number of clinical and pathological features with Dementia with Lewy bodies (DLB). The present study was done to examine whether or not plasma annexin A5 is a specific marker for AD, when being compared with the levels of DLB patients. As Apolipoprotein E (ApoE) gene subtype ε4 (ApoE-ε4) has been noticed as the probable genetic factor for AD, we also examined and compared ApoE genotype in both AD and DLB. METHODS: Blood samples were obtained from 150 patients with AD (aged 77.6 ± 6.5 years), 50 patients of DLB (79.4 ± 5.0) and 279 community-dwelling healthy elderly individuals of comparable age and sex (75.6 ± 8.1). All AD patients met NINCDS-ADRDA criteria and all DLB patients were diagnosed as probable DLB according to the latest consensus diagnostic criteria. Quantification was done using the Chemiluminescent Enzyme Immunoassay (CLEIA) Technique (SphereLight assay) using the monoclonal antibodies against annexin A5. DNA genotyping of ApoE was performed by distinguishing unique combinations of Hha1 fragments of PCR-amplified genomic DNA products. RESULTS: The plasma level of annexin A5 was significantly higher in AD patients than in the healthy individuals (control) (P < 0.0001). The plasma annexin A5 level was also significantly higher in DLB patients than in the control group (P < 0.0001). From the ROC curves with plasma annexin A5 concentrations, the mean areas under the curve were 0.863 and 0.838 for the AD/control and DLB/control, respectively. The rate of ApoE4 carrier status and the frequency of the ε4 allele were significantly higher in AD or DLB than in control and there was no significant difference between AD and DLB. CONCLUSIONS: These results suggest that both annexin A5 and ApoE4 are common markers for AD and DLB.

Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain.

BACKGROUND: X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid ß-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent. RESULTS: To confirm the interaction of X11L with human APP ectopically expressed in mouse brain, we examined the amyloidogenic metabolism of human APP in two lines of human APP transgenic mice generated to also lack X11L. In agreement with previous reports from our lab and others, we found that the amyloidogenic metabolism of human APP increased in the absence of X11L. CONCLUSION: X11L appears to aid in the suppression of amyloidogenic processing of human APP in brain in vivo, as has been demonstrated by previous studies using several human APP transgenic lines with various genetic backgrounds. X11L appears to regulate human APP in a manner similar to that seen in endogenous mouse APP metabolism.

Changes in localization of cytochrome P450 cholesterol side-chain cleavage (P450scc) in Japanese eel testis and ovary during gonadal development.

In this study, we generated and characterized a polyclonal antiserum against eel P450 cholesterol side-chain cleavage (P450scc) using a recombinant protein as the antigen. We examined the localization and abundance of P450scc by immunohistochemistry in Japanese eel testes and ovaries during artificially induced gonadal development. P450scc mRNA localization was also examined by in situ hybridization. In male eels, testicular development was induced by a single injection of human chorionic gonadotropin (HCG). In females, ovarian development was induced by weekly injections of salmon pituitary homogenate (SPH). Before HCG injection, the testis contained germ cells that were primarily type A spermatogonia. Additionally, several clusters of immunoreactive cells for P450scc were localized in the interstitial Leydig cells, but no P450scc mRNA signals were detected. This suggests that P450scc is either a relatively stable protein or it is produced by a mRNA that is present at too low a level to detect. Shortly after a single injection of HCG, expression of P450scc mRNA was stimulated and the number of immunoreactive clusters and their staining intensity were both increased. P450scc mRNA fell to an undetectable level 3 days after hormonal stimulation. Although the P450scc protein also decreased at the same time as the mRNA, it remained at a detectable level throughout this period. P450scc mRNA, but not the P450scc protein, was also detected in the spermatids and spermatozoa. The biological significance of P450scc mRNA expression at this stage is unknown. Prior to experimentation, the ovary contained oocytes that were developed to the oil-droplet stage, with several clusters of immunoreactive cells localized in the thecal layer and ovigerous lamella epithelium. Expression of P450scc mRNA was also stimulated by SPH injections in the ovary. In contrast to the testis, P450scc mRNA was continuously detected in the thecal cell layer throughout artificially induced maturation, possibly due to a repeated stimulus by the SPH injection every week. Clusters of immunoreactive cells in the thecal cell layer increased in number as ovarian development progressed. This increase in P450scc mRNA and protein may explain, at least in part, the increase in serum steroid hormones in female eels. The P450scc antiserum clearly immunostained interrenal steroidogenic cells in the head kidney of not only eel but also goldfish, indicating that this antibody could also be used in other teleost species.