Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6 and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G(2)/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G(2) damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein. In this article we studied the effect of methionine oxidation on the conformation of recombinant human growth hormone (r-hGH). In literature it is reported that oxidation of methionine residues induces conformation changes on r-hGH. In our study, oxidation of r-hGH was performed by incubation with hydrogen peroxide under mild conditions. Mass spectrometry and chromatographic analysis revealed that oxidation with hydrogen peroxide resulted in more than 90% of oxidized r-hGH. By extensive spectroscopic characterizations no detectable change in conformation and aggregation of r-hGH after oxidation was found. In conclusion, mild oxidation conditions led to selective oxidation of the two more accessible methionine residues of r-hGH (Met(14) and Met(125)) and did not results in any conformation change of the protein. These findings prove that oxidation of human growth hormone does not influence protein conformation and demonstrate the importance of employing mild conditions during production of oxidized protein. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:110-122, 2011

Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Fasciola hepatica: time-dependent disruption of spermatogenesis following in vivo treatment with triclabendazole

Sheep infected with the Cullompton isolate of Fasciola hepatica were treated with triclabendazole at a concentration of 10 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the liver and, where present, from the gall bladder at 48, 72 and 96 h post-treatment (pt). Gross changes to the spermatogenic cells of the testis were examined by histology and ultrastructural alterations were visualised via transmission electron microscopy. Disruption was progressive in nature, with the testis tubules becoming shrunken, vacuolated and gradually more denuded of cellular content over the 96-h time period. From 48 h pt, the number of primary and secondary spermatogonia decreased and multinucleate spermatogonial cells were frequent. Later, developmental stages were uncommon, giving rise to much empty space within the tubules. By 72 h pt, the tubules contained many apoptotic and degraded cells and had an extremely disorganised appearance. At 96 h pt, the tubules were almost completely empty, with the exception of the remains of degraded spermatogenic cells. These results indicate that triclabendazole severely disrupts spermatogenesis in the liver fluke from 48 h pt in vivo.

Carbon Isotopic Fractionation of CFCs during Abiotic and Biotic Degradation

Carbon stable isotope ((13)C) fractionation in chlorofluorocarbon (CFC) compounds arising from abiotic (chemical) degradation using zero-valent iron (ZVI) and biotic (landfill gas attenuation) processes is investigated. Batch tests (at 25 °C) for CFC-113 and CFC-11 using ZVI show quantitative degradation of CFC-113 to HCFC-123a and CFC-1113 following pseudo-first-order kinetics corresponding to a half-life (t(1/2)) of 20.5 h, and a ZVI surface-area normalized rate constant (k(SA)) of -(9.8 ± 0.5) × 10(-5) L m(-2) h(-1). CFC-11 degraded to trace HCFC-21 and HCFC-31 following pseudo-first-order kinetics corresponding to t(1/2) = 17.3 h and k(SA) = -(1.2 ± 0.5) × 10(-4) L m(-2) h(-1). Significant kinetic isotope effects of e(‰) = -5.0 ± 0.3 (CFC-113) and -17.8 ± 4.8 (CFC-11) were observed. Compound-specific carbon isotope analyses also have been used here to characterize source signatures of CFC gases (HCFC-22, CFC-12, HFC-134a, HCFC-142b, CFC-114, CFC-11, CFC-113) for urban (UAA), rural/remote (RAA), and landfill (LAA) ambient air samples, as well as in situ surface flux chamber (FLUX; NO FLUX) and landfill gas (LFG) samples at the Dargan Road site, Northern Ireland. The latter values reflect biotic degradation and isotopic fractionation in LFG production, and local atmospheric impact of landfill emissions through the cover. Isotopic fractionations of ?(13)C ~ -13‰ (HCFC-22), ?(13)C ~ -35‰ (CFC-12) and ?(13)C ~ -15‰ (CFC-11) were observed for LFG in comparison to characteristic solvent source signatures, with the magnitude of the isotopic effect for CFC-11 apparently similar to the kinetic isotope effect for (abiotic) ZVI degradation.

Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: Regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.

Acid-catalyzed hydrolysis of cellulose and cellulosic waste using a microwave reactor system

A microwave reactor system was investigated as a potential technique to maximize sugar yield for the hydrolysis of municipal solid waste for ethanol production. Specifically, dilute acid hydrolysis of a-cellulose and waste cellulosic biomass (grass clippings) with phosphoric acid was undertaken within the microwave reactor system. The experimental data and reaction kinetic analysis indicate that the use of a microwave reactor system can successfully facilitate dilute acid hydrolysis of cellulose and waste cellulosic biomass, producing high yields of total sugars in short reaction times. The maximum yield of reducing sugars was obtained at 7.5% (w/v) phosphoric acid and 160 degrees C, corresponding to 60% of the theoretical total sugars, with a reaction time of 5 min. When using a very low acid concentration (0.4% w/v) for the hydrolysis in the microwave reactor, it was found that 10 g of total sugars/100 g dry mass was produced, which is significant considering the low acid concentration. When hydrolyzing grass clippings using the microwave reactor, the optimum conditions were an acid concentration of 2.5% (w/v), 175 degrees C with a 15 min reaction time, giving 18 g/100 g dry mass of total sugars, with xylose being the sugar with the highest yield. It was observed that pentose sugars were more easily formed but also more easily degraded, these being significantly affected by increases in acid concentration and temperature. Kinetic modeling of the data indicated that the use of microwave heating may account for an increase in reaction rate constant, k(1), found in this study in comparison with conventional systems described in the literature.

Simple method for the rapid simultaneous screening of photocatalytic activity over multiple positions of self-cleaning films

An intelligent ink, previously shown to be capable of rapidly assessing photocatalytic activity, was simply applied via a felt-pen onto a commercially available piece of Activ (TM) self-cleaning glass. The ink, comprising of redox dye resazurin and the sacrificial electron donor glycerol within an aqueous hydroxy ethyl cellulose (HEC) polymer media, was photocatalytically degraded in a two-step process. The key initial stage was the photo-reductive conversion of resazurin to resorufin, whereby a colour change from blue to pink occurred. The latter stage was the subsequent photo-reduction of the resorufin, where a slower change from pink to colourless was seen. Red and green components of red-green-blue colour extracted from flat-bed scanner digital images of resazurin ink coated photocatalytic films at intervals during the photocatalysis reaction were inversely proportional to the changes seen via UV-visible absorption spectroscopy and indicative of reaction kinetics. A 3 x 3 grid of intelligent ink was drawn onto a piece of Activ (TM) and a glass blank. The photocatalysis reaction was monitored solely by flat-bed digital scanning. Red-green-blue values of respective positions on the grid were extracted using a custom-built program entitled RGB Extractor (c). The program was capable of extracting a number of 5 x 5 pixel averages of red-green-blue components simultaneously. Allocation of merely three coordinates allowed for the automatic generation of a grid, with scroll-bars controlling the number of positions to be extracted on the grid formed. No significant change in red and green components for any position on the glass blank was observed; however, the Activ (TM) film displayed a homogenous photo-reduction of the dye, reaching maxima in red and minima in green components in 23 +/- 3 and 14 +/- 2 min, respectively. A compositionally graded N-doped titania film synthesised in house via a combinatorial APCVD reaction was also photocatalytically tested by this method where 247 positions on a 13 x 19 grid were simultaneously analysed. The dramatic variation in photocatalysis observed was rapidly quantified for all positions (2-3 hours) allowing for correlations to be made between thicknesses and N : Ti% compositions attained from Swanepoel and WDX analysis, respectively. N incorporation within this system was found to be detrimental to film activity for the photocatalysis reaction of intelligent ink under 365 nm light.

Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB -13064

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C-10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C-14:0, C-16:0, C-16:1, and C-18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF2-alpha-VHL Axis as the Major Regulator of Erythropoietin Production

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.

ELECTRON-ENERGY DISTRIBUTION-FUNCTIONS AND NEGATIVE-ION CONCENTRATIONS IN TANDEM AND HYBRID MULTICUSP NEGATIVE HYDROGEN-ION SOURCES

The second derivative of a Langmuir probe characteristic is used to establish the electron energy distribution function (EEDF) in both a tandem and hybrid multicusp H- ion source. Moveable probes are used to establish the spatial variation of the EEDF. The negative ion density is measured by laser induced photo-detachment. In the case of the hybrid source the EEDF consists of a cold Maxwellian in the central region of the source; the electron temperature increases with increasing discharge current (rising from 0.3 eV at 1 A to 1.2 eV at 50 A when the pressure is 0.4 Pa). A hot-electron tail exists in the EEDF of the driver region adjacent to each filament which is shown to consist of a distinct group of primary electrons at low pressure (0.08 Pa) but becomes degraded mainly through inelastic collisions at higher pressures (0.27 Pa). The tandem source, on the other hand, has a single driver region which extends throughout the central region. The primary electron confinement times are much longer so that even at the lowest pressure considered (0.07 Pa) the primaries are degraded. In both cases the measured EEDF at specific locations and values of discharge operating parameters are used to establish the rate coefficients for the processes of importance in H- production and destruction.

Holocene climate change, vegetation history and human impact in the Central Mediterranean: evidence from the Maltese Islands

Holocene climates and human impact in the Mediterranean basin have received much attention, but the Maltese Islands in the Central Mediterranean, although a pivotal area, have been little researched. Here, sedimentary and palynological data are presented for three cores from the Holocene coastal and shallowmarine
deposits of the Maltese Islands. These show deforestation from Pinus-Cupressaceae woodland in the early Neolithic, and then a long, but relatively stable history of agriculturally degraded environments to the present day. The major climate events which have affected the Italian and Balkan peninsulas to the
north, and Tunisia to the south, are not reflected in the pollen diagrams from the Maltese Islands because of the strong anthropogenic imprint on the Maltese vegetation from early in the Neolithic. Previous suggestions of environmentally-driven agricultural collapse at the end of the Neolithic appear, however,
to be substantiated and may be linked to regional aridification around 4300 cal. BP. Depopulation in early Medieval times is not supported by the current palynological evidence.

Brain activity during ankle proprioceptive stimulation predicts balance performance in young and older adults

Proprioceptive information from the foot/ankle provides important information regarding body sway for balance control, especially in situations where visual information is degraded or absent. Given known increases in catastrophic injury due to falls with older age, understanding the neural basis of proprioceptive processing for balance control is particularly important for older adults. In the present study, we linked neural activity in response to stimulation of key foot proprioceptors (i.e., muscle spindles) with balance ability across the lifespan. Twenty young and 20 older human adults underwent proprioceptive mapping; foot tendon vibration was compared with vibration of a nearby bone in an fMRI environment to determine regions of the brain that were active in response to muscle spindle stimulation. Several body sway metrics were also calculated for the same participants on an eyes-closed balance task. Based on regression analyses, multiple clusters of voxels were identified showing a significant relationship between muscle spindle stimulation-induced neural activity and maximum center of pressure excursion in the anterior-posterior direction. In this case, increased activation was associated with greater balance performance in parietal, frontal, and insular cortical areas, as well as structures within the basal ganglia. These correlated regions were age- and foot-stimulation side-independent and largely localized to right-sided areas of the brain thought to be involved in monitoring stimulus-driven shifts of attention. These findings support the notion that, beyond fundamental peripheral reflex mechanisms, central processing of proprioceptive signals from the foot is critical for balance control.

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57-63%) as opposed to albumen, while 71-80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low µg kg -1 concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.

Degradation of 4-fluorobiphenyl by mycorrhizal fungi as determined by (19)F nuclear magnetic resonance spectroscopy and (14)C radiolabelling analysis

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using (19)F nuclear magnetic resonance (NMR) spectroscopy in combination with (14)C radioisotope-detected high-performance liquid chromatography ((14)C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. (14)C-HPLC profiles indicated that there were four major biotransformation products, whereas (19)F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4'-ol and 4-fluorobiphen-3'-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.