A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.     

Programmed death-1 gene polymorphism (PD-1.5 C/T) is associated with colon cancer

Programmed death-1 (PD-1), expressed by activated T cells, is a negative regulator of T lymphocytes. The associations of the immune response-related genes with cancer have been demonstrated. In this study, the PD-1.5 C/T (+7785) polymorphism was investigated in 200 colorectal cancer patients and 200 healthy individuals as controls by nested polymerase chain reaction-restriction fragment length polymorphism method. The genotype and allele frequencies at PD-1.5 position were not significantly different between control individuals and the overall colorectal cancer patients. However, subdivision of the patients by the location (175 colon cancer and 25 rectal cancer) revealed a significant difference between colon cancer patients and healthy individuals (p=0.026), and between colon and rectal cancer patients (p=0.017). The frequency of the CT genotype was significantly higher in colon cancer patients than in control individuals (58.3% vs. 44.8%, Bonferroni corrected p-value=0.024; OR=1.74; 95% CI=1.15-2.62), and in rectal cancer patients (58.3% vs. 28.0%, Bonferroni corrected p-value=0.012; OR=3.59; 95% CI=1.42-9.04). Characteristics of the patients including age, sex, tumor grade and stage were not associated with the PD-1.5 polymorphism. Our results show a significant association between PD-1.5 polymorphism and colon cancer. Larger numbers of patients are required to investigate comprehensively the association of rectal cancer with PD-1.5 polymorphism.     

MicroRNA-125b Down-regulates Matrix Metallopeptidase 13 and Inhibits Cutaneous Squamous Cell Carcinoma Cell Proliferation, Migration, and Invasion

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.     

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca²⁺ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr⁶⁹⁷ and/or Thr⁸⁵⁵ (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser⁶⁹⁶ prevents phosphorylation at Thr⁶⁹⁷. However, the effects of Ser⁸⁵⁴ and dual Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵), Ser phosphorylation events (Ser⁶⁹⁶/Ser⁸⁵⁴) and dual Ser/Thr phosphorylation events (Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵). Dual phosphorylation at Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr⁶⁹⁷ and Thr⁸⁵⁵ by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵ in rat caudal artery, whereas U46619 induced Thr⁶⁹⁷ and Thr⁸⁵⁵ phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.     

Water wires in atomistic models of the Hv1 proton channel

The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Antioxidant therapies in traumatic brain and spinal cord injury

Free radical formation and oxidative damage have been extensively investigated and validated as important contributors to the pathophysiology of acute central nervous system injury. The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is an early event following injury occurring within minutes of mechanical impact. A key component in this event is peroxynitrite-induced lipid peroxidation. As discussed in this review, peroxynitrite formation and lipid peroxidation irreversibly damages neuronal membrane lipids and protein function, which results in subsequent disruptions in ion homeostasis, glutamate-mediated excitotoxicity, mitochondrial respiratory failure and microvascular damage. Antioxidant approaches include the inhibition and/or scavenging of superoxide, peroxynitrite, or carbonyl compounds, the inhibition of lipid peroxidation and the targeting of the endogenous antioxidant defense system. This review covers the preclinical and clinical literature supporting the role of ROS and RNS and their derived oxygen free radicals in the secondary injury response following acute traumatic brain injury (TBI) and spinal cord injury (SCI) and reviews the past and current trends in the development of antioxidant therapeutic strategies. Combinatorial treatment with the suggested mechanistically complementary antioxidants will also be discussed as a promising neuroprotective approach in TBI and SCI therapeutic research. This article is part of a Special Issue entitled: Antioxidants and antioxidant treatment in disease.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.