Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.     

β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase,UCH-L1

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.     

Splenic hypertrophy and extramedullary hematopoiesis induced in male Syrian hamsters by short photoperiod or melatonin injections and reversed by melatonin pellets or pinealectomy

Adult male Syrian hamsters either placed in a short photoperiod alone or kept in a long photoperiod and given daily afternoon injections of the pineal indole melatonin (25 micrograms) exhibited splenic hypertrophy and extramedullary hematopoiesis in addition to a marked regression in testicular weight. The testicular regression as well as the changes in spleen weight and histology could be prevented if the animals in short photoperiod were either pinealectomized or implanted subcutaneously with a pellet containing 1 mg melatonin. Female Syrian hamsters given afternoon injections of melatonin for 7 or 12 weeks had ovaries devoid of corpora lutea; additionally, these animals had reduced relative spleen weights compared to the control animals. In conclusion, it is apparent that spleen weight varies with the functional status of the gonads. Splenic hypertrophy accompanied by pineal-induced testicular regression in males may be related to splenic extramedullary hematopoiesis.     

Intra-tree foraging behavior of Ceratitis capitata flies in relation to host fruit density and quality

We examined the intra-tree foraging behavior of individually-released, wild-population Mediterranean fruit flies (medflies), Ceratitis capitata (Wiedemann), on field-caged host trees bearing each of three different densities (0, 3, or 12 per tree) of non-infested host fruit (kumquat) or each of two levels of fruit quality (12 non-infested fruit or 12 fruit infested with eggs and covered with host marking pheromone). With increasing density of non-infested fruit, medflies tended to remain longer in trees, visit more fruit before leaving, oviposit more often, accept a proportionately smaller number of fruit visited, and emigrate sooner after the last egg was laid (i.e. have a shorter Giving-Up-Time). Medflies spent much less time, oviposited much less often, and exhibited a longer Giving-Up-Time on trees harboring pheromone-marked fruit than non-infested fruit. Variation in temperature within the range at which experiments were conducted (25–36°C) had little detectable influence on foraging behavior. We compare our findings with published findings on the intra-tree foraging behavior of another tephritid fly, Rhagoletis pomonella (Walsh), and with current foraging behavior theory. We discuss implications of our findings with respect to medfly management strategies, particularly fruit stripping in eradication programs and use of synthetic marking pheromone for control.

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Résumé Nous avons étudié le comportement de prospection dans un arbre, de femelles d'une population sauvage de C. capitata, libérées individuellement à l'intérieur de cages contenant des Eriobotrya japonica (kumquat), portant chacun 3 densités différentes de fruits no contaminés (0, 3, 12 par arbre) et chacun 2 niveaux de qualité de fruits: 12 fruits non infestés ou 12 fruits contaminés par des oeufs et recouverts de phéromone de marquage de l'hôte. C. capitata avait terndance à rester plus longtemps dans les arbres, à visiter plus de fruits avant le quitter, à pondre plus souvent, à accepter proportionnellement un nombre plus réduit de fruits déjà visités, à émigrer plus tôt après la ponte du dernier oeuf (c'est-à-dire à présenter un temps d'abandon plus bref), quand la densité des fruits non contaminés augmentait. C. capitata a dépensé beaucoup moins de temps, pondu beaucoup moins souvent, et présenté un temps d'abandon plus long sur les arbres portant des fruits marqués par la phéromone que sur ceux ayant des fruits non contaminés. Les variations de température dans la gamme de cells où les observations ont eu lieu (23–36°C) n'ont eu qu'une faible influence décelable sur le comportement de prospection. Nous avons comparé nos résultats avec ceux publiés sur la prospection à l'intérieur de l'arbre par une autre téphritide (Rhagoletis pomonella) et avec la théorie dominante sur le comportement de prospection. Nous discutons les conséquences de nos résultats sur les stratégies de lutte contre C. capitata, en particulier l'élimination des fruits dans les plans d'erradication et l'utilisation de phéromone synthétique de marquage.

     

Cellular location of the cleavage event of the polymeric immunoglobulin receptor and fate of its anchoring domain in the rat hepatocyte.

Transcytosis of polymeric immunoglobulin (pIg) across glandular and mucosal epithelia is mediated by a member of the immunoglobulin supergene family, the pIg receptor. During transcellular routing, the receptor is cleaved and its ectoplasmic domain, known as secretory component (SC), is released into secretions bound to pIg. Using receptor-domain-specific antibodies, we have combined cell fractionation and immunoblotting of rat liver to examine the cellular routing of the receptor, the cellular location of the cleavage event and the fate of the anchor domain. Cleavage is a late event in receptor processing. It appears to occur at the canalicular plasma membrane, since intact receptor is present in this membrane domain and no SC is detected in whole liver homogenate or in cell fractions. The membrane anchor remaining after cleavage can be recovered in bile, as well as in a low-density fraction obtained after equilibrium centrifugation of liver (microsomal fractions) on sucrose density gradients. These data suggest that the membrane-anchor domain may be internalized as well as secreted together with SC into bile.     

The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Str?hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.