Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

AHR,a novel acute hypoxia‐response sequence,drives reporter gene expression under hypoxia in vitro and in vivo

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter‐driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia‐response sequence)‐GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP‐expressing vector under the control of CMV (cytomegalovirus promoter‐GFP). We transduced AHR‐GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV‐GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR‐GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR‐GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia‐activated gene expression system.     

mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes

Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.     

Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells

Clearance of apoptotic cells is necessary for tissue development, homeostasis and resolution of inflammation. The uptake of apoptotic cells is initiated by an 'eat-me' signal, such as phosphatidylserine, on the cell surface and phagocytes recognize the signal by using specific receptors. In this study, we show that the soluble form of the receptor for advanced glycation end products (RAGE) binds to phosphatidylserine as well as to the apoptotic thymocytes. RAGE-deficient (Rage(-/-)) alveolar macrophages showed impaired phagocytosis of apoptotic thymocytes and defective clearance of apoptotic neutrophils in Rage(-/-) mice. Our results indicate that RAGE functions as a phosphatidylserine receptor and assists in the clearance of apoptotic cells.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.     

Impaired deformability of erythrocytes in diabetic rat and human: investigation by the nickel-mesh-filtration technique

Comprehensive research to quantify the deformability of erythrocytes in diabetic animals and humans has been lacking. The objective of this study was to compare the impairment of erythrocyte deformability in diabetic rats and patients by use of the same rheologic method. Deformability was investigated in streptozotocin-induced diabetic rats and diabetic patients, by using the highly sensitive and quantitative nickel-mesh-filtration technique. Erythrocyte filterability (whole-cell deformability) was defined as flow rate of hematocrit-adjusted erythrocyte suspension relative to that of saline (%). Hematological and biochemical data for diabetic rats did not differ from those for age-matched control rats except for hyperglycemia and malnutrition. Erythrocyte filterability for diabetic rats was significantly lower than that for control rats (69.4 ± 10.1%, n = 8, compared with 83.1 ± 4.2%, n = 8; p < 0.001). Likewise, erythrocyte filterability for diabetic patients was significantly impaired compared with that for controls (87.6 ± 3.4%, n = 174, compared with 88.6 ± 2.1%, n = 51; p = 0.046). Stepwise multiple regression analysis revealed that this impairment was mostly attributable to associated obesity (BMI, p = 0.029) and glycemic stress (HbA1c(JDS), p = 0.046). We therefore conclude that erythrocyte filterability is commonly impaired in diabetic rats and in humans. Moreover, metabolic risk accumulation further impairs erythrocyte filterability, resulting in derangement of the microcirculation.     

Validity of self-reported cancer among a Japanese population: recent results from a population-based prospective study in Japan (JPHC Study)

The recent tendency of Japanese towards greater acceptance of being informed that they have cancer, along with the growing understanding and use of informed consent, appears to have improved the accuracy of self-reported cancer. To clarify the recent validity of self-reports, we measured the sensitivity and positive predictive value of self-reported cancer among a Japanese population. Using a 10-year follow-up questionnaire conducted in 2000-2004 and the cancer registry of the JPHC Study cohort (n=93,680), we calculated the sensitivity and positive predictive value of self-reported cancer diagnoses over 10 years. Sensitivity and positive predictive value of total self-reported cancer diagnoses were 53% and 60%, respectively, but varied by site, at 62% and 52% for stomach, 38% and 47% for colorectum, 57% and 46% for lung, 34% and 31% for liver, 82% and 58% for breast, and 59% and 22% for uterus, respectively. Sensitivity was considerably improved from that in the previous report (36%), which tested for 1990-1995, but was still not considered satisfactory. Self-reported diagnoses of cancer do not provide sufficient accuracy for the detection and classification of incident cancers. Our findings may be extrapolated to other Japanese populations.