Faculty Bibliography

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway

INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.

INTRODUCTIONThe generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.

The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer

Neuronal survival requires continuous lysosomal turnover of cellular constituents delivered by autophagy and endocytosis. Primary lysosomal dysfunction in inherited congenital 'lysosomal storage' disorders is well known to cause severe neurodegenerative phenotypes associated with accumulations of lysosomes and autophagic vacuoles (AVs). Recently, the number of inherited adult-onset neurodegenerative diseases caused by proteins that regulate protein sorting and degradation within the endocytic and autophagic pathways has grown considerably. In this Perspective, we classify a group of neurodegenerative diseases across the lifespan as disorders of lysosomal function, which feature extensive autophagic-endocytic-lysosomal neuropathology and may share mechanisms of neurodegeneration related to degradative failure and lysosomal destabilization. We highlight Alzheimer's disease as a disease within this group and discuss how each of the genes and other risk factors promoting this disease contribute to progressive lysosomal dysfunction and neuronal cell death

OBJECTIVE: Subjects with diabetes experience an increased risk of myocardial infarction and cardiac failure compared with nondiabetic age-matched individuals. The receptor for advanced glycation end products (RAGE) is upregulated in diabetic tissues. In this study, we tested the hypothesis that RAGE affected ischemia/reperfusion (I/R) injury in the diabetic myocardium. In diabetic rat hearts, expression of RAGE and its ligands was enhanced and localized particularly to both endothelial cells and mononuclear phagocytes. RESEARCH DESIGN AND METHODS: To specifically dissect the impact of RAGE, homozygous RAGE-null mice and transgenic (Tg) mice expressing cytoplasmic domain-deleted RAGE (DN RAGE), in which RAGE-dependent signal transduction was deficient in endothelial cells or mononuclear phagocytes, were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to I/R. RESULTS: Diabetic RAGE-null mice were significantly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH and lower glycoxidation products carboxymethyl-lysine (CML) and pentosidine, improved functional recovery, and increased ATP. In diabetic Tg mice expressing DN RAGE in endothelial cells or mononuclear phagocytes, markers of ischemic injury and CML were significantly reduced, and levels of ATP were increased in heart tissue compared with littermate diabetic controls. Furthermore, key markers of apoptosis, caspase-3 activity and cytochrome c release, were reduced in the hearts of diabetic RAGE-modified mice compared with wild-type diabetic littermates in I/R. CONCLUSIONS: These findings demonstrate novel and key roles for RAGE in I/R injury in the diabetic heart

Activation of nuclear factor-kappaB (NF-kappaB), a key feature of the neurotrophin signaling, has been shown to be critical for neuronal survival under pathologic settings. However, the precise mechanism by which neurotrophins activate NF-kappaB is not well understood. Here we report that the Ankyrin-rich Membrane Spanning (ARMS/Kidins220) protein, a novel transmembrane substrate of tropomyosin receptor kinase B (TrkB), plays an important role in NF-kappaB signaling elicited by brain-derived neurotrophic factor (BDNF). Accordingly, depletion of ARMS by specific RNA interference, or disruption of ARMS-TrkB interaction with expression of dominant-negative ARMS mutant, abolished BDNF-induced signaling to NF-kappaB. Our data further suggests that ARMS may promote NF-kappaB signaling via activation of mitogen-activated kinase (MAPK) and IkappaB kinase (IKK), thereby facilitating phosphorylation of RelA (major NF-kappaB subunit) at an IKK-sensitive site. The results shown here identify ARMS as a major factor that links neurotrophin signaling to NF-kappaB

Health-care safety management has recently been highlighted for patient safety. However, specialist-based risks in clinical settings have hardly been discussed in Japan so far. A review of dermatological legal claims may delineate these risks. This study examined court precedents from the databases "Courts in Japan" and LEX/DB. Thirty-four dermatology-related civil cases were found from 1968-2006. Of the 34 cases, 32 (94%) were judged and two (6%) were retried. Of these 32 cases, 11 (34%) were appealed to higher courts. Among the 34 litigations, the defendants of eight (23%) were dermatology specialists, 20 (59%) were non-dermatologists and six (18%) of unknown specialty. The defendants' negligence was determined at either level in court in 25 of the 34 cases. The negligence in these 25 cases was categorized into five groups: (i) delayed diagnosis (none); (ii) complication during diagnosis procedure (one, 4%); (iii) inappropriate treatment (nine, 36%); (iv) complication during treatment procedure (10, 40%); and (v) insufficient informed consent (five, 20%). The present study may help to improve strategies for health-care safety management in the dermatological field in Japan.

The conventional method for producing embryonic stem (ES) cell-derived knockout or transgenic mice involves injection of ES cells into normal, diploid blastocysts followed by several rounds of breeding of resultant chimeras and thus is a time-consuming and inefficient procedure. F0 ES cell pups can also be derived directly from tetraploid embryo complementation, which requires fusion of two-cell embryos. Recently, F0 ES cell pups have been produced by injection of ES cells into eight-cell embryos using a laser-assisted micromanipulation system. We report a simple method for producing F0 ES cell germline-competent mice by piezo injection of ES cells into four- or eight-cell embryos. The efficiency of producing live, transgenic mice by this method is higher than that with the tetraploid blastocyst complementation method. This efficient and economical technique for directly producing F0 ES cell offspring can be applicable in many laboratories for creating genetically manipulated mice using ES cell technology and also for stringent testing of the developmental potency of new ES cell or other types of pluripotent stem cell lines