Unique Phosphorylation of Protein Kinase C-α in PC12 Cells Induces Resistance to Translocation and Down-regulation

Cell exposure to phorbol ester stimulates translocation and activation of protein kinase C (PKC), ultimately followed by its down-regulation. Upon activation, PKC-alpha, the best studied isotype of the PKC family, undergoes changes in its phosphorylation state. With a two-dimensional immunoblot procedure we have previously shown the existence in PC12 cells of several multiply phosphorylated forms of PKC-alpha, whose number increases in response to phorbol esters (Gatti, A., Wang, X., and Robinson, P. J. (1996) Biochim. Biophys. Acta 1313, 111-118). Using the same experimental system, here we report that besides the predominant pool of 80-kDa PKC-alpha forms that respond to phorbol ester by translocating to the cell membranes and down-regulating, there is a small pool of cytosolic 82-kDa PKC-alpha forms that are characterized by a more acidic pI and by an unique resistance to phorbol ester-mediated translocation and down-regulation. The appearance of similarly slower migrating and more acidic PKC-alpha forms is reproduced upon in vitro autophosphorylation in the presence of phosphatidylserine and phorbol ester, but not in the presence of calcium. These results suggest that site-specific transphosphorylation or autophosphorylation of this kinase may regulate its subcellular localization and susceptibility to down-regulation

Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

<p>Abstract</p> <p>Background</p> <p>The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown.</p> <p>Results</p> <p>We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.</p> <p>Conclusion</p> <p>Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.</p

Ignition Characterization Test Results for the LO2/Ethanol Propellant Combination

A series of contracts were issued by the Marshall Space Flight Center (MSFC) of the National Aeronautics and Space Administration (NASA) under the auspices of the Exploration Systems Mission Directorate to develop and expand the maturity of candidate technologies considered to be important for future space exploration. One such technology was to determine the viability of incorporating non-toxic propellants for Reaction Control Subsystems (RCS). Contract NAS8-01109 was issued to Aerojet to develop a dual thrust Reaction Control Engine (RCE) that utilized liquid oxygen and ethanol as the propellants. The dual thrust RCE incorporated a primary thrust level of 870 lbf, and a vernier thrust level of 10 - 30 lbf. The preferred RCS approach for the dual thrust RCE was to utilize pressure-fed liquid oxygen (LOX) and ethanol propellants; however, previous dual thrust feasibility testing incorporated GOX/Ethanol igniters as opposed to LOX/Ethanol igniters in the design. GOX/Ethanol was easier to ignite, but this combination had system design implications of providing GOX for the igniters. A LOX/Ethanol igniter was desired; however, extensive LOX/Ethanol ignition data over the anticipated operating range for the dual thrust RCE did not exist. Therefore, Aerojet designed and tested a workhorse LOX igniter to determine LOX/Ethanol ignition characteristics as part of a risk mitigation effort for the dual thrust RCE design. LOX, encompassing potential two-phase flow conditions anticipated being present in real mission applications. A workhorse igniter was designed to accommodate the hll LOX design flowrate, as well as a reduced GOX flowrate. It was reasoned that the initial LOX flow through the igniter would flash to GOX due to the latent heat stored in the hardware, causing a reduced oxygen flowrate because of a choked, or sonic, flow condition through the injection elements. As LOX flow continued, the hardware would chill-in, with the injected oxygen flow transitioning from cold GOX through two-phase flow to subcooled LOX. permitted oxygen state points to be determined in the igniter oxidizer manifold, and gas-side igniter chamber thermocouples provided chamber thermal profile characteristics. The cold flow chamber pressure (P(sub c)) for each test was determined and coupled with the igniter chamber diameter (D(sub c)) to calculate the characteristic quench parameter (P(sub c) x D(sub c)), which was plotted as a function of core mixture ratio, m. Ignition limits were determined over a broad range of valve inlet conditions, and ignition was demonstrated with oxygen inlet conditions that ranged from subcooled 210 R LOX to 486 R GOX. Once ignited at cold GOX conditions, combustion was continuous as the hardware chilled in and the core mixture ratio transitioned from values near 1.0 to over 12.5. Pulsing is required in typical RCS engines; therefore, the workhorse igniter was pulse tested to verify the ability to provide the required ignition for a pulsing RCE. The minimum electrical pulse width (EPW) of the dual thrust RCE was 0.080 seconds

Identification of TPIT and other novel autoantigens in lymphocytic hypophysitis; immunoscreening of a pituitary cDNA library and development of immunoprecipitation assays

BACKGROUND: Lymphocytic hypophysitis is an organ-specific autoimmune disease of the pituitary gland. A specific and sensitive serological test currently does not exist to aid in the diagnosis. OBJECTIVE: To identify target autoantigens in lymphocytic hypophysitis and develop a diagnostic assay for these proteins. DESIGN/METHODS: A pituitary cDNA expression library was immunoscreened using sera from four patients with lymphocytic hypophysitis. Relevant cDNA clones from screening, along with previously identified autoantigens pituitary gland-specific factor 1a and 2 (PGSF1a and PGSF2) and neuron-specific enolase (NSE) were tested in an in vitro transcription and translation immunoprecipitation assay. The corticotroph-specific transcription factor, TPIT, was investigated separately as a candidate autoantigen. RESULTS: Significantly positive autoantibody reactivity against TPIT was found in 9/86 hypophysitis patients vs 1/90 controls (P = 0.018). The reactivity against TPIT was not specific for lymphocytic hypophysitis with autoantibodies detectable in the sera from patients with other autoimmune endocrine diseases. Autoantibodies were also detected against chromodomain-helicase-DNA binding protein 8, presynaptic cytomatrix protein (piccolo), Ca(2+)-dependent secretion activator, PGSF2 and NSE in serum samples from patients with lymphocytic hypophysitis, but at a frequency that did not differ from healthy controls. Importantly, 8/86 patients with lymphocytic hypophysitis had autoantibodies against any two autoantigens in comparison with 0/90 controls (P = 0.0093). CONCLUSIONS: TPIT, a corticotroph-specific transcription factor, was identified as a target autoantigen in 10.5% of patients with lymphocytic hypophysitis. Further autoantigens related to vesicle processing were also identified as potential autoantigens with different immunoreactivity patterns in patients and controls

Novel rat tail discitis model using bioluminescent Staphylococcus aureus

Management of spondylodiscitis is a challenging clinical problem requiring medical and surgical treatment strategies. The purpose of this study was to establish a rat model of spondylodiscitis that utilizes bioluminescent Staphylococcus aureus, thus permitting in-vivo surveillance of infection intensity. Inocula of the bioluminescent S. aureus strain XEN36 were created in concentrations of 102 CFU/0.1 mL, 104 CFU/0.1 mL, and 106 CFU/0.1 mL. Three groups of rats were injected with the bacteria in the most proximal intervertebral tail segment. The third most proximal tail segment was injected with saline as a control. Bioluminescence was measured at baseline, 3 days, and weekly for a total of 6 weeks. Detected bioluminescence for each group peaked at day three and returned to baseline at 21 days. The average intensity was highest for the experimental group injected with the most concentrated bacterial solution (106 CFU/0.1 mL). Radiographic analysis revealed loss of intervertebral disc space and evidence of osseous bridging. Saline injected spaces exhibited no decrease in intervertebral spacing as compared to distal sites. Histologic analysis revealed neutrophilic infiltrates, destruction of the annulus fibrosus and nucleus pulposus, destruction of vertebral endplates, and osseous bridging. Saline injected discs exhibited preserved annulus fibrosus and nucleus pulposus on histology. This study demonstrates that injection of bioluminescent S. aureus into the intervertebral disc of a rat tail is a viable animal model for spondylodiscitis research. This model allows for real-time, in-vivo quantification of infection intensity, which may decrease the number of animals required for infection studies of the intervertebral disc

Human Streptococcus agalactiae Isolate in Nile Tilapia (Oreochromis niloticus)

Streptococcus agalactiae, the Lancefield group B streptococcus (GBS) long recognized as a mammalian pathogen, is an emerging concern with regard to fish. We show that a GBS serotype Ia multilocus sequence type ST-7 isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia (Oreochromis niloticus)

In situ structure of an intact lipopolysaccharide-bound bacterial surface layer

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications

Carbon dioxide and ocean acidification observations in UK waters. Synthesis report with a focus on 2010–2015

Key messages: 1.1 The process of ocean acidification is now relatively well-documented at the global scale as a long-term trend in the open ocean. However, short-term and spatial variability can be high. 1.2 New datasets made available since Charting Progress 2 make it possible to greatly improve the characterisation of CO2 and ocean acidification in UK waters. 3.1 Recent UK cruise data contribute to large gaps in national and global datasets. 3.2 The new UK measurements confirm that pH is highly variable, therefore it is important to measure consistently to determine any long term trends. 3.3 Over the past 30 years, North Sea pH has decreased at 0.0035±0.0014 pH units per year. 3.4 Upper ocean pH values are highest in spring, lowest in autumn. These changes reflect the seasonal cycles in photosynthesis, respiration (decomposition) and water mixing. 3.5 Carbonate saturation states are minimal in the winter, and lower in 7 more northerly, colder waters. This temperature-dependence could have implications for future warming of the seas. 3.6 Over the annual cycle, North-west European seas are net sinks of CO2. However, during late summer to autumn months, some coastal waters may be significant sources. 3.7 In seasonally-stratified waters, sea-floor organisms naturally experience lower pH and saturation states; they may therefore be more vulnerable to threshold changes. 3.8 Large pH changes (0.5 - 1.0 units) can occur in the top 1 cm of sediment; however, such effects are not well-documented. 3.9 A coupled forecast model estimates the decrease in pH trend within the North Sea to be -0.0036±0.00034 pH units per year, under a high greenhouse gas emissions scenario (RCP 8.5). 3.10 Seasonal estimates from the forecast model demonstrate areas of the North Sea that are particularly vulnerable to aragonite undersaturation

A mass spectrometry-based approach to distinguish annular and specific lipid binding to membrane proteins

Membrane proteins engage in a variety of contacts with theirsurrounding lipids, but distinguishing between specifically boundlipids, and non-specific annular interactionsis a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the Presenilin Homologue protease are subject to constant exchange with detergent. Bycontrast,detergent-resistantlipids bound at the dimer interface in the Leucine transportershowdecreased koffrates in molecular dynamics simulations.Turning tothe lipid flippase MurJ, we findthat addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergentfromthe highly protected active site.In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution. [Abstract copyright: © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.