MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.     

Site-specific effects of peptide lipidation on beta-amyloid aggregation and cytotoxicity

Beta-amyloid (Abeta) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of Abeta by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified Abeta at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-Abeta, K28-OA-Abeta, and Nalpha-OA-Abeta. Octanoylation of these sites on Abeta-(1-40) had strikingly different effects on fibril formation. K16-OA-Abeta and K28-OA-Abeta, but not Nalpha-OA-Abeta, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-Abeta peptides to cross-seed solutions of Abeta was the inverse of their ability to form fibrils on their own (i.e. Abeta approximately Nalpha-OA-Abeta>K16-OA-Abeta>K28-OA-Abeta). By CD spectroscopy, K16-OA-Abeta and K28-OA-Abeta had increased beta-sheet propensity compared with Abeta-(1-40) or Nalpha-OA-Abeta. K16-OA-Abeta and K28-OA-Abeta were more amphiphilic than Abeta-(1-40) or Nalpha-OA-Abeta, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-Abeta and K28-OA-Abeta are much more cytotoxic to N2A cells than Abeta-(1-40) or Nalpha-OA-Abeta. The greater cytotoxicity of K16-OA-Abeta and K28-OA-Abeta may reflect their greater amphiphilicity. We conclude that lipidation can make Abeta more prone to aggregation and more cytotoxic, but these effects are highly site-specific.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Bacterial chemotaxis transverse to axial flow in a microfluidic channel

Swimming bacteria sense and respond to chemical signals in their environment. Chemotaxis is the directed migration of a bacterial population toward increasing concentrations of a chemical that they perceive to be beneficial to their survival. Bacteria that are indigenous to groundwater environments exhibit chemotaxis toward chemical contaminants such as hydrocarbons, which they are also able to degrade. This phenomenon may facilitate bioremediation processes by bringing bacteria into closer proximity to these contaminants. A microfluidic device was assembled to study chemotaxis transverse to advective flow. Using a T-shaped channel design (T-sensor), two fluid streams were brought into contact by impinging flow. They then flowed adjacent to each other along a transparent channel. An advantage to this design is that it allows real-time visualization of bacterial distributions within the channel. Under laminar flow conditions a chemotactic driving force was created perpendicular to the direction of flow by diffusion of the chemical attractant from one input stream to the other. A comparison of the chemotactic band behavior in the absence and presence of flow showed that fluid velocity did not significantly impede chemotactic migration in the transverse direction.     

Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland

Ghrelin (G-HH) synthesized in several tissues including salivary and stomach glands stimulates appetite in humans by modulating neuropeptide Y neurons in the hypothalamic arcuate nucleus. Loss of appetite is one of the most important symptoms of stomach cancer. We conducted a study using immunohistochemistry to determine whether salivary glands and stomach cancer tissues produce ghrelin. We determined that negative ghrelin immunohistochemistry discriminates tumors from normal tissues and may therefore further our understanding of the clinically important problem of reduced food intake and anorexia in cancer patients. Radioimmunoassay analyses confirmed that cancer cells do not produce a G-HH peptide, whereas normal cells yield this peptide.