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The efficacy of auxinic herbicides, a valuable weed control tool for growers worldwide, has been shown to vary with the time of day in which applications are made. However, little is known about the mechanisms causing this phenomenon. Investigating the differential in planta behavior of these herbicides across different times of application may grant an ability to advise which properties of auxinic herbicides are desirable when applications must be made around the clock. Radiolabeled herbicide experiments demonstrated a likely increase in ATP-binding cassette subfamily B (ABCB)-mediated 2,4-D and dicamba transport in Palmer amaranth (Amaranthus palmeri S. Watson) at simulated dawn compared to mid-day, as dose response models indicated that many orders of magnitude higher concentrations of N-1-naphthylphthalamic acid (NPA) and verapamil, respectively, are required to inhibit translocation by 50% at simulated sunrise compared to mid-day. Gas chromatographic analysis displayed that ethylene evolution in A. palmeri was higher when dicamba was applied during mid-day compared to sunrise. Furthermore, it was found that inhibition of translocation via 2,3,5-triiodobenzoic acid (TIBA) resulted in an increased amount of 2,4-D-induced ethylene evolution at sunrise, and the inhibition of dicamba translocation via NPA reversed the difference in ethylene evolution across time of application. Dawn applications of these herbicides were associated with increased expression of a putative 9-cis-epoxycarotenoid dioxygenase biosynthesis gene NCED1, while there was a notable lack of trends observed across times of day and across herbicides with ACS1, encoding 1-aminocyclopropane-1-carboxylic acid synthase. Overall, this research indicates that translocation is differentially regulated via specific protein-level mechanisms across times of application, and that ethylene release, a chief phytotoxic process involved in the response to auxinic herbicides, is related to translocation. Furthermore, transcriptional regulation of abscisic acid involvement in phytotoxicity and/or translocation are suggested., Competing Interests: No authors have competing interests

Harpin proteins stimulate hypersensitive response (HR) in plants. However, the mechanism by which HR is regulated is not clear. The role of the auxin, indole-3-acetic acid (IAA), in the control of harpin-stimulated HR was investigated. IAA was used to inhibit HR that was stimulated by purified fusion harpin(Xoo) protein in tobacco. Semi-quantitative PCR and qRT-PCR were employed to detect the expression of HR related genes. IAA at 100 μM reversed harpin-induced HR which was inhibited by 500 μM 2,3,5-triiodobenzoic acid (TIBA). Semi-quantitative PCR and qRT-PCR showed the combined application of 100 μM IAA and harpin protein from Xanthomonas oryzae enhanced the expression of HR marker gene, hsr203J, but weakened the expression of the disease-defense gene, chia5. TIBA also decreased the expression of hsr203J but increased the expression of chia5. Thus, the auxin can reverse harpin(Xoo)-induced HR.

Background: The accumulation of macrophages in inflamed atherosclerotic plaques has long been recognized. In an attempt to develop an imaging agent for detection of vulnerable plaques, we evaluated the feasibility of a liposomal-iodine nanoparticle contrast agent for computed tomography imaging of macrophage-rich atherosclerotic plaques in a mouse model., Methods and Results: Liposomal-iodine formulations varying in particle size and polyethylene glycol coating were fabricated and shown to stably encapsulate the iodine compound. In vitro uptake studies using optical and computed tomography imaging in the RAW 264.7 macrophage cell line identified the formulation that promoted maximal uptake. Dual-energy computed tomography imaging using this formulation in apolipoprotein E-deficient (ApoE(-/-)) mice (n=8) and control C57BL/6 mice (n=6) followed by spectral decomposition of the dual-energy images enabled imaging of the liposomes localized in the plaque. Imaging cytometry confirmed the presence of liposomes in the plaque and their colocalization with a small fraction (≈2%) of the macrophages in the plaque., Conclusions: The results demonstrate the feasibility of imaging macrophage-rich atherosclerotic plaques using a liposomal-iodine nanoparticle contrast agent and dual-energy computed tomography.

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

PARsylation [poly(ADP-ribosyl)ation] of proteins is implicated in the regulation of diverse physiological processes. Tankyrase is a molecular scaffold with this catalytic activity and has been proposed as a regulator of vesicular trafficking on the basis, in part, of its Golgi localization in non-polarized cells. Little is known about tankyrase localization in polarized epithelial cells. Using MDCK (Madin-Darby canine kidney) cells as a model, we found that E-cadherin-mediated intercellular adhesion recruits tankyrase from the cytoplasm to the lateral membrane (including the tight junction), where it stably associates with detergent-insoluble structures. This recruitment is mostly completed within 8 h of calcium-induced formation of cell-cell contact. Conversely, when intercellular adhesion is disrupted by calcium deprivation, tankyrase returns from the lateral membrane to the cytoplasm and becomes more soluble in detergents. The PARsylating activity of tankyrase promotes its dissociation from the lateral membrane as well as its ubiquitination and proteasome-mediated degradation, resulting in an apparent protein half-life of approximately 2 h. Inhibition of tankyrase autoPARsylation using H2O2-induced NAD+ depletion or PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride] treatment results in tankyrase stabilization and accumulation at the lateral membrane. By contrast, stabilization through proteasome inhibition results in tankyrase accumulation in the cytoplasm. These data suggest that cell-cell contact promotes tankyrase association with the lateral membrane, whereas PARsylating activity promotes translocation to the cytosol, which is followed by ubiquitination and proteasome-mediated degradation. Since the lateral membrane is a sorting station that ensures domain-specific delivery of basolateral membrane proteins, the regulated tankyrase recruitment to this site is consistent with a role in polarized protein targeting in epithelial cells.

5-Amino-2,4,6-triiodoisophthalic acid (ATIA), both a precursor and a degradative intermediate of triiodinated contrast media, was anaerobically converted by sludge from a wastewater treatment plant. ATIA conversion took place only when an electron donor such as ethanol was added. A stable mixed culture was established by transfer to a defined synthetic mineral medium with ATIA and ethanol. It could be maintained for 1 year when the sulfate concentration was kept below 30 microM. Transient appearance of 5-amino-2,4-diiodoisophthalic acid, iodide release (2.7 mol iodide/mol ATIA) and accumulation of 5-aminoisophthalic acid indicated that ATIA was reductively dehalogenated. The enriched mixed culture also dehalogenated ATIA derivatives but deiodination remained incomplete. ATIA was the sole terminal electron acceptor used by the mixed culture during deiodination. The ratio of electrons transferred to ATIA, 0.83, was consistent with a respiratory metabolism. Formate, acetate, lactate, butyrate and hydrogen were also used as electron donors. Deiodination was inhibited by a headspace of air or by addition of nitrate, sulfite or thiosulfate. The reaction was 2.6 times slower with sulfate than without.

Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter, an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

Rationale and Objectives: Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol., Materials and Methods: Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically., Results: Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%., Conclusion: In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.

The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.

Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.

Iopromide (Schering, Berlin) is a new nonionic, monomeric contrast medium containing three different substituents on the triiodinated benzene ring. Iopromide exhibits low osmolality and viscosity in aqueous solutions of high concentrations. It has been shown to have a remarkably low intravenous toxicity in mice and rats. Neural tolerance was found to be equal to or better than that of metrizamide when injected in rats intracisternally and intracerebrally, respectively. The effects of iopromide after selective peripheral and cerebral arterial injections in rats were demonstrated to be very moderate at high dosages. The interaction of iopromide with proteins and membranes was found to be considerably low due to its hydrophilicity. Excretion of iopromide is fast and predominantly by the renal route. On the basis of the preclinical profile iopromide is a very promising contrast agent, being most suitable for all angiographic indications, including digital subtraction angiography, urography, and computed tomography.

The absorption of the nonionic contrast medium iohexol, the clearance tracer 51Cr-ethylenediaminetetraacetic acid, and the blood-pool marker 125I-human serum albumin was studied after subarachnoid and subdural injection in rabbits. Subdural deposition of the contrast medium and 51Cr-ethylenediaminetetraacetic acid resulted in a faster absorption rate and higher achieved blood levels than a subarachnoid injection of the two substances, where a slow absorption to lower blood concentrations was observed. No significant differences in absorption rate could be shown after subdural and subarachnoid administration of iodine-labelled albumin. The excretion of iohexol was observed for 1 week after the intrathecal injection. For both the subdural and subarachnoid depositions, about 83% of the injected iohexol was found in urine within 24 hr after injection. The total recovery of iohexol after 1 week was 96% (range, 87%-101%).

The magnitude and time course of contrast enhancement in spontaneous canine brain tumors was determined for two contrast agents: meglumine iothalamate and sodium meglumine ioxaglate. Tumor enhancement during contrast infusion and at 5, 10, 15, 30, and 45 min was measured using quantitative computed tomography. Blood iodine was measured using x-ray fluorescence. Peak contrast enhancement occurred during the infusion, and the magnitude was the same for both agents. Per gram of iodine infused, blood iodine was 12.4% higher with ioxaglate than iothalamate. The monoionic dimer ioxaglate is as effective as iothalamate for enhancement of canine brain tumors.

Four new myelographic agents, metrizamide, iopamidol, iohexol, and iotrol, were studied in the subarachnoid space of cynomolgus monkeys. Plain films and computed tomographic scans documented the transport of each material throughout the space and into the brain. At the concentration used (300 mg I/ml), all gave good radiopacity for myelography and delineation of the cerebral subarachnoid space. All four cleared similarly from the ventricular system. Metrizamide, however, penetrated the brain in greater degree and persisted longer than the other three agents. Next in persistence was iopamidol and least, and both statistically equal, iotrol and iohexol.