Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

Purification and Characterization of a Lectin with High Hemagglutination Property Isolated from <Emphasis Type="Italic">Allium altaicum</Emphasis>

A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was ~24 kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues pre-proprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57–94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity.     

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.     

Selective matrix metalloproteinase inhibition attenuates progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure

Matrix metalloproteinases (MMPs) contribute to the progression of left ventricular (LV) dysfunction and remodeling associated with heart failure (HF). The present study examined the long-term effects of a selective MMP inhibitor PG-530742 (PG) on the progression of LV dysfunction and remodeling in dogs with HF. Chronic HF [LV ejection fraction (LVEF), 相似文献   

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.     

PCNA promotes processive DNA end resection by Exo1

Exo1-mediated resection of DNA double-strand break ends generates 3′ single-stranded DNA overhangs required for homology-based DNA repair and activation of the ATR-dependent checkpoint. Despite its critical importance in inducing the overall DNA damage response, the mechanisms and regulation of the Exo1 resection pathway remain incompletely understood. Here, we identify the ring-shaped DNA clamp PCNA as a new factor in the Exo1 resection pathway. Using mammalian cells, Xenopus nuclear extracts and purified proteins, we show that after DNA damage, PCNA loads onto double-strand breaks and promotes Exo1 damage association through direct interaction with Exo1. By tethering Exo1 to the DNA substrate, PCNA confers processivity to Exo1 in resection. This role of PCNA in DNA resection is analogous to its function in DNA replication where PCNA serves as a processivity co-factor for DNA polymerases.     

Neural Regulation of Muscle Glucose 6-Phosphate Dehydrogenase: Effect of Batrachotoxin and Tetrodotoxin

Abstract: We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) activity in the rat skeletal muscle is regulated by putative axonally derived neurotrophic factors. This was accomplished by comparing the effects of nerve section and subperineural injection of batrachotoxin (BTX) or tetrodotoxin (TTX) on G6PD in rat extensor digitorum longus (EDL) muscle. BTX, an agent known to block nerve impulse conduction and axonal transport, increased G6PD activity to 155% and 163% of control by days 2 and 4 after injection. Denervation of the EDL muscle by section of the peroneal nerve 10–20 mm from its entrance to the muscle caused G6PD activity to increase to 170% of control by day 1 and to 200% and 180% of control by days 2 and 4, respectively. The increase in enzyme activity after denervation and after subperineural injection of BTX was due in part to muscle inactivity resulting from blockade of nerve impulses. This conclusion is based upon the observation that subperineural injection of TTX at an identical site in the peroneal nerve caused a small but significant (30%) increase in G6PD activity after 4 days. Choline acetyltransferase (CAT) activity was assessed as a measure of the efficacy of blockade of slow axonal transport. Decreases in CAT activity following denervation or injection of BTX or TTX were parallel to increases in G6PD activity observed under these conditions. These results argue for a role of axonal transport in neural regulation of muscle G6PD, with a small contribution by neuromuscular activity.