Forced unfolding of proteins within cells

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding.     

Accelerated uplift and magmatic intrusion of the Yellowstone caldera, 2004 to 2006

The Yellowstone caldera began a rapid episode of ground uplift in mid-2004, revealed by Global Positioning System and interferometric synthetic aperture radar measurements, at rates up to 7 centimeters per year, which is over three times faster than previously observed inflation rates. Source modeling of the deformation data suggests an expanding volcanic sill of approximately 1200 square kilometers at a 10-kilometer depth beneath the caldera, coincident with the top of a seismically imaged crustal magma chamber. The modeled rate of source volume increase is 0.1 cubic kilometer per year, similar to the amount of magma intrusion required to supply the observed high heat flow of the caldera. This evidence suggests magma recharge as the main mechanism for the accelerated uplift, although pressurization of magmatic fluids cannot be ruled out.     

Crystal structure of the malaria vaccine candidate apical membrane antigen 1

Apical membrane antigen 1 from Plasmodium is a leading malaria vaccine candidate. The protein is essential for host-cell invasion, but its molecular function is unknown. The crystal structure of the three domains comprising the ectoplasmic region of the antigen from P. vivax, solved at 1.8 angstrom resolution, shows that domains I and II belong to the PAN motif, which defines a superfamily of protein folds implicated in receptor binding. We also mapped the epitope of an invasion-inhibitory monoclonal antibody specific for the P. falciparum ortholog and modeled this to the structure. The location of the epitope and current knowledge on structure-function correlations for PAN domains together suggest a receptor-binding role during invasion in which domain II plays a critical part. These results are likely to aid vaccine and drug design.     

Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis

The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.     

Bioaccumulation of melamine in catfish muscle following continuous, low-dose, oral administration

In this study, catfish muscle was analyzed for melamine (MEL) and cyanuric acid (CYA) residues following experimental feeding with low doses of MEL and MEL and CYA (MEL+CYA) and with the insoluble melamine-cyanurate complex (MEL=CYA). Catfish were daily fed 0.1 mg/kg BW of MEL for 15, 28, or 42 days, 0.1 mg/kg BW of MEL+CYA for 28 days, 2.5 mg/kg BW of MEL+CYA for 14 days, or 400 mg/kg BW of MEL=CYA for 3 days. Residues in the tissue were determined by LC-MS/MS. MEL was extracted with acidic acetonitrile, followed by defatting with dichloromethane, and isolated with cation exchange solid phase extraction (SPE). For CYA analysis, fish were extracted with dilute acetic acid, defatted with hexane, and cleaned up with a graphitic carbon SPE. Catfish fed 0.1 mg/kg BW of MEL reached a maximum muscle residue concentration of 0.33 ± 0.04 mg/kg (ppm) after 28 days of continuous feeding. The same concentration was found for MEL+CYA feeding at the 0.1 mg/kg BW level for 28 days. Feeding at 2.5 mg/kg BW of MEL+CYA yielded muscle concentrations above the 2.5 mg/kg level of concern for most of the study fish. Finally, catfish fed high levels of the MEL=CYA complex (400 mg/kg BW) accumulated relatively little MEL in the muscle (0.14 ± 0.07 mg/kg) and, unlike treatment with MEL+CYA, did not form renal melamine-cyanurate crystals. Appreciable concentrations of CYA were not detected in any of the muscles tested. These studies provide data to model the bioaccumulation of triazine residues into edible fish tissue as a result of the continuous consumption of adulterated feed.     

Denitrification potential of intermittently saturated floodplain soils from a subtropical perennial stream and an ephemeral tributary

Denitrification has the potential to remove excess nitrogen from groundwater passing through riparian buffers, thus improving water quality downstream. In regions with markedly seasonal precipitation, transient stream flow events may be important in saturating adjacent floodplain soils and intermittently providing the anaerobic conditions necessary for denitrification to occur. In two experiments we characterised the denitrification potential of soils from two contrasting floodplains that experience intermittent saturation. We quantified under controlled laboratory conditions: 1) potential rates of denitrification in these soils with depth and over time, for a typical period of saturation; and 2) the influences on rates of nitrate and organic carbon. Treatments differed between experiments, but in each case soil-water slurries were incubated anaerobically with differing amendments of organic carbon and nitrate; denitrification rates were measured at selected time intervals by the acetylene-block technique; and slurry filtrates were analysed for various chemical constituents. In the first experiment (ephemeral tributary), denitrification was evident in soils from both depths (0-0.3 m; 0.3-1.1 m) within hours of saturation. Before Day 2, mean denitrification rates at each depth were generally comparable, irrespective of added substrates; mean rates (Days 0 and 1) were 5.2 ± 0.3 mg N kg dry soil⁻¹ day⁻¹ (0-0.3 m) and 1.6 ± 0.2 mg N kg dry soil⁻¹ day⁻¹ (0.3-1.1 m). Rates generally peaked on Days 2 or 3. The availability of labile organic carbon was a major constraint on denitrification in these soils. Acetate addition greatly increased rates, reaching a maximum in ephemeral floodplain soils of 17.4 ± 1.8 mg N kg dry soil⁻¹ day⁻¹ on Day 2: in one deep-soil treatment (low nitrate) this overcame differences in rates observed with depth when acetate was not added, although the rate increase in the other deep-soil treatment (high nitrate) was significantly less (P ≤ 0.01). Without acetate, peak denitrification rates in this experiment were 6.9 ± 0.4 and 2.8 ± 0.2 mg N kg dry soil⁻¹ day⁻¹ in surface and deep soils, respectively. Differences in rates were observed with depth on all occasions, despite similar initial concentrations of dissolved organic carbon (DOC) at both depths. Levels of substrate addition in the second experiment (perennial stream) more closely reflected natural conditions at the site. Mean denitrification rates were consistently much higher in surface soil (P ≤ 0.001), while the source of water used in the slurries (surface water or groundwater from the site) had little effect on rates at any depth. Mean rates when all treatments retained nitrate were: 4.5 ± 0.3 mg N kg dry soil⁻¹ day⁻¹ (0-0.3 m depth); 0.8 ± 0.3 mg N kg dry soil⁻¹ day⁻¹ (0.3-1.0 m); and 0.6 ± 0.1 mg N kg dry soil⁻¹ day⁻¹ (1.8-3.5 m). For comparable treatments and soil depths, denitrification potentials at both sites were similar, apart from higher initial rates in the ephemeral floodplain soils, probably associated with their higher DOC content and possibly also their history of more frequent saturation. The rapid onset of denitrification and the rates measured in these soils suggest there may be considerable potential for nitrate removal from groundwater in these floodplain environments during relatively short periods of saturation.     

Formation of Maillard reaction products during heat treatment of carrots

As indicators of the early stage of the Maillard reaction in carrots, N-(furoylmethyl) amino acids (FMAAs) formed during acid hydrolysis of the corresponding Amadori products were analyzed using RP-HPLC with UV detection. N(ε)-FM-Lys (furosine), FM-Gly, FM-Ala, FM-Val, FM-Ile, FM-Leu, and FM-GABA were identified using synthesized standard material by means of mass spectrometry. Furthermore, N(ε)-carboxymethyllysine (CML) and pyrraline were analyzed as indicators for advanced stages of glycation. For commercial samples with high water content, the formation of Amadori compounds predominates, whereas the advanced stage of Maillard reaction plays only a minor part. Carrot juices, baby food, and tinned carrots showed quite low rates of amino acid modification up to 5%. For dehydrated carrots, significantly higher values for Amadori products were measured, corresponding to a lysine derivatization of up to 58% and nearly 100% derivatization of GABA. Drying experiments revealed great differences in reactivity between the amino acids studied. Whereas furosine reached constant values quite quickly, some FMAAs showed a continuous increase with heating time, indicating that selected FMAAs can be used as a hallmark for the early Maillard reaction to control processing conditions.     

Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.     

Hemolytic anemia and induction of phase II detoxification enzymes by diprop-1-enyl sulfide in rats: dose-response study

Epidemiological evidence indicates that a high dietary intake of plants of the Allium family, such as garlic and onions, is associated with a decreased risk of cancer in humans. It has been suggested that this chemopreventative effect involves the ability of the aliphatic sulfides derived from these vegetables to increase tissue activities of phase II detoxification enzymes. Several highly effective inducers from garlic have been identified, but most of the previously studied compounds from onion have proved to be only weakly active. In the present study, the inductive activity of another onion-derived sulfide, diprop-1-enyl sulfide, has been investigated. This substance was a potent inducer of phase II enzymes in rats, showing significant effects in the lungs and in the lower part of the gastrointestinal tract, suggesting that diprop-1-enyl sulfide could be a useful chemopreventative agent at these sites. At high dose levels, diprop-1-enyl sulfide caused hemolytic anemia, which may be due to in vivo conversion of the sulfide to active metabolites.