Genetic mutation analysis in Japanese patients with non-syndromic congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect occurring in humans and some transcriptional factors have been identified as causative. However, additional mutation analysis of these genes is necessary to develop effective diagnostic and medical treatment methods. We conducted sequence analysis of the coding regions of NKX2.5, GATA4, TBX1, TBX5, TBX20, CFC1 and ZIC3 in 111 Japanese patients with non-syndromic CHD and 9 of their relatives. All patient samples were also analyzed by multiplex ligation-dependent probe amplification using probes involved in chromosome deletion related to CHD. Five novel variations of TBX5, GATA4 and TBX20 were detected in 6 of the patients, whereas none were found in 200 controls. The TBX5 variation p.Pro108Thr, located in the T-box domain, was identified in a patient with tricuspid atresia, an exon-intron boundary variation of GATA4 (IVS4+5G>A) was detected in a Tetralogy of Fallot patient and an 8p23 microdeletion was detected in one patient with atrioventricular septal defect and psychomotor delay. A total of seven non-synonymous polymorphisms were found in the patients and controls. Accumulation of novel variations of genes involving the cardiac development may be required for better understanding of CHD.

Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice.

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin ß1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-ß1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.

Differential proteomic shotgun analysis elucidates involvement of water channel aquaporin 8 in presence of α-amylase in the colon.

Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC-MS alignment based approach, we compared wild-type and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of α-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in α-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating α-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts.

Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia.

Transient forebrain ischemia induces calpain-mediated degradation of the neuronal cytoskeleton, alpha-fodrin, and this results in ischemic neuronal death. In this study, we investigated the spatial distribution and temporal changes of calpain-catalyzed alpha-fodrin proteolysis in focal cerebral ischemia and examined the effects of a calpain inhibitor. Ischemia was induced in gerbils by 3-h middle cerebral artery occlusion followed by reperfusion. Animals were divided into four groups: a sham-operated group, an ischemic group, a vehicle-treated group, and a calpain inhibitor-treated group. Intravenous injections of vehicle or calpain inhibitor I were administered 30 min before ischemia. Infarct volumes were measured 1 day after reperfusion and the spatial distribution of calpain-catalyzed alpha-fodrin proteolysis was investigated by immunohistochemistry 15 min, 1 h, 4 h, and 1 day after reperfusion. Infarct volume (mean +/- SD) in the ischemic group and the vehicle-treated group was 204.6 +/- 19.1 mm3 and 212.4 +/- 16.3 mm3, respectively, and the calpain inhibitor I reduced the infarct volume [149.4 +/- 25.2 mm3 (P < 0.05)]. Immunoblot analysis demonstrated that calpain inhibitor reduced proteolysis. Ischemia induced fodrin proteolysis in the ischemic core and the peri-infarct zone within 15 min after reperfusion, with proteolysis developing quickly in the ischemic core and more slowly in the peri-infarct zone. Proteolysis preceded neuronal death in the peri-infarct zone. Calpain inhibitor I ameliorated neuronal death in the peri-infarct zone but not in the ischemic core. Thus, calpain plays a pivotal role on focal ischemia as well as in global ischemia.

Calpain induces proteolysis of neuronal cytoskeleton in ischemic gerbil forebrain.

We investigated the relationship between the activity of calcium-dependent protease (calpain) and the ischemic neuronal damage. We also investigated the mechanism of ischemic resistance in astrocytes. In gerbil, a 10-min forebrain ischemia was induced by occlusion of both common carotid arteries. The calpain-induced proteolysis of cytoskeleton (fodrin) was examined by immunohistochemistry. Immunolocalization of micro and m-calpain was also examined. Intact fodrin was observed both in neurons and astrocytes, but proteolyzed fodrin was not observed in normal brain. Fifteen minutes after ischemia, proteolysis of fodrin took place in putamen, parietal cortex and hippocampal CA1. The proteolysis extended to thalamus 4 h after ischemia after which the immunoreactivity faded down in all areas except hippocampus. On day 7, the proteolysis was still observed only in hippocampus. Neurons with the proteolysis of soma resulted in neuronal death. Throughout the experiment, the proteolysis was not observed in astrocytes. micro -Calpain was observed only in neurons but m-calpain was observed both in neurons and astrocytes. The ischemia induced only micro -calpain activation, which resulted in fodrin proteolysis of neurons with differential spatial distribution and temporal course. The proteolysis was developed rapidly and was completed within 24 h in all vulnerable regions except hippocampal CA1. The proteolysis preceded the neuronal death. The mechanism of the proteolysis seemed to be involved by Ca(2+) influx via glutamate receptor and rapid neuronal death seemed reasonable. The reason why neuronal death in CA1 evolved slowly was not clarified. In astrocytes, fodrin was not proteolyzed by m-calpain. The low Ca(2+)-sensitivity of m-calpain may be the reason of ischemic resistance in astrocytes.