Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes

Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.     

Inhibitory effects of an antisense oligonucleotide in an experimentally infected mouse model of influenza A virus

The antiviral effects of a 20-mer antisense phosphorothioate oligonucleotide, PB2-as, on influenza A virus infection in mice were examined and compared to those of PB2-as encapsulated with several cationic liposomes. Intravenous injection of PB2-as, as a complex with DMRIE-C, a cationic liposome, was most effective for prolonging the mean survival time in days (MSDs) and increasing the survival rates of mice infected with the influenza A virus. In addition, the liposomal PB2-as significantly inhibited viral growth in lung tissues. These results suggest that PB2-as encapsulated with DMRIE-C may be active against the influenza A virus infection through the inhibition of virus replication in the mouse lung.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

Chewing number is related to incremental increases in body weight from 20 years of age in Japanese middle‐aged adults

doi: 10.1111/j.1741‐2358.2012.00666.x Chewing number is related to incremental increases in body weight from 20 years of age in Japanese middle‐aged adults Background: Eating habits are associated with both current obesity and incremental increases in body weight from young adulthood, but no study has focused on chewing number during meals among community residents. Objective: This study focused on the relationship between chewing number and incremental increases in body weight from 20 years of age. Methods: A total of 93 persons aged 35–61 years participated. The subjects were asked to set the device and record their chewing number during each meal on a particular day. They were also asked whether their body weight had increased by 10 kg or more since they were 20 years old. Results: The body weight of 28 subjects (30%) had increased more than 10 kg since the age of 20 years. Total chewing number showed a relationship with such body weight increases. The odds ratio of weight increments of more than 10 kg for the lowest tertile group was 4.6 [95% confidence interval (CI), 1.3–16.2] relative to the highest tertile group (Model 1). The odds ratio of weight increments for the lowest tertile group increased to 6.3 (95% CI, 1.6–25.4) in Model 2 and to 9.1 (95% CI, 1.7–49.8) in Model 3. Conclusion: Although this study was limited because it did not consider all risk factors, categorical chewing number was related independently to body weight increments of more than 10 kg from 20 years of age.     

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at −70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 μM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.     

Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres

Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature-sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S-phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C-terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2-Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.     

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.