Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Plasma membrane of a murine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins.

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase. T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

The position of localized soil compaction determines root and subsequent shoot growth responses

Plants growing in soils typically experience a mixture of loose and compact soil. The hypothesis that the proportion of a root system exposed to compact soil and/or the timing at which this exposure occurs determines shoot growth responses was tested. Broccoli (Brassica oleracea var. italica cv. Greenbelt) seedlings were grown in pot experiments with compact, loose and localized soil compaction created by either horizontal (compact subsoils 75 or 150 mm below loose topsoil) or vertical (adjacent compact and loose columns of soil) configurations of loose (1.2 Mg m(-3)) and compact (1.8 Mg m(-3)) soil. Entirely compact soil reduced leaf area by up to 54%, relative to loose soil. When compaction was localized, only the vertical columns of compact and loose soil reduced leaf area (by 30%). Neither the proportion of roots in compact soil nor the timing of exposure could explain the differing shoot growth responses to localized soil compaction. Instead, the strong relationship between total root length and leaf area (r(2)=0.92) indicated that localized soil compaction reduced shoot growth only when it suppressed total root length. This occurred when isolated root axes of the same plant were exposed to vertical columns of compact and loose soil. When a single root axis grew through loose soil into either a shallow or deep compact subsoil, compensatory root growth in the loose soil maintained total root length and thus shoot growth was unaffected. These contrasting root systems responses to localized soil compaction may explain the variable shoot growth responses observed under heterogeneous conditions.     

Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm

Bran from bread wheat (Triticum aestivum ‘Babbler’) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.Wheat grain (Triticum aestivum) is a major cereal crop and staple food in many parts of the world. The endosperm is the main nutritional component and is extracted in milling to produce base ingredients such as flour and semolina. Crop yield and quality may be compromised by both environmental and biological stresses. Wheat varieties are known to vary in their resistance to such stresses, probably due to individual differences in defense protein levels (Demeke and Morris, 2002; Bonnin et al., 2005; Yarullina et al., 2005). Cereal grain contains many defense proteins that have been categorized according to their mode of action and structural similarities. A major class of these is the pathogenesis-related (PR) proteins, which include PR-1, PR-2 (β -1,3-glucanases), PR-3 (chitinases), PR-4 (wheatwin1), and PR-5 (thaumatin-like proteins; Selitrennikoff, 2001; Desmond et al., 2006). Other known defense proteins are xylanase inhibitor proteins (XIPs) and α -amylase inhibitor proteins (Mundy et al., 1984; Payan et al., 2003). All of these defense proteins have both general and specific roles that contribute to plant survival, although little is known of their location within the various grain tissues, particularly the multiple layers that constitute bran.Proteomic analysis of wheat grain has previously been applied to identify proteins in the germ and endosperm (Skylas et al., 2000; Wong et al., 2004; Mak et al., 2006), but analysis of bran and bran tissue fractions has not been reported. Collection of sufficiently pure bran tissue fractions has limited progress, mainly due to the strong bonds between the various bran tissue layers and endosperm in dry grain. Thus, a method to obtain bran layers free from contaminants, such as adjacent tissue and endosperm, is required to provide a sample suitable for proteomic analysis. Soaking whole grain in water causes the endosperm to soften, allowing it to be easily removed and washed from the bran; the bran becomes malleable enough to dissect. While this approach might not identify the proteome of dry grain fractions, it is the best available representation of the three distinct tissue fractions in grains, namely the outer layer (epidermis and hypodermis), intermediate layer (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells; Antoine et al., 2003, 2004). Using this method, water-soluble proteins that diffuse from the grain can be collected and identified.In this study we aimed (1) to dissect bran into the three separate tissue fractions described above and to identify the protein complement of each fraction using proteomics, (2) to confirm the location of three major defense proteins identified (one from each microfraction) using immunolocalization, and (3) to identify water-soluble proteins and assay any defense-related proteins for enzymatic activity.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Growth dynamics of mechanically impeded lupin roots: does altered morphology induce hypoxia?

BACKGROUND AND AIMS: Root axes elongate slowly and swell radially under mechanical impedance. However, temporal and spatial changes to impeded root apices have only been described qualitatively. This paper aims (a) to quantify morphological changes to root apices and (b) assess whether these changes pre-dispose young root tissues to hypoxia. METHODS: Lupin (Lupinus angustifolius) seedlings were grown into coarse sand that was pressurized through a diaphragm to generate mechanical impedance on growing root axes. In situ observations yielded growth rates and root response to hypoxia. Roots were then removed to assess morphology, cell lengths and local growth velocities. Oxygen uptake into excised segments was measured. KEY RESULTS: An applied pressure of 15 kPa slowed root extension by 75% after 10-20 h while the same axes thickened by about 50%. The most terminal 2-3 mm of axes did not respond morphologically to impedance, in spite of the slower flux of cells out of this region. The basal boundary of root extension encroached to within 4 mm of the apex (cf. 10 mm in unimpeded roots), while radial swelling extended 10 mm behind the apex in impeded roots. Oxygen demand by segments of these short, thick, impeded roots was significantly different from segments of unimpeded roots when the zones of elongation in each treatment were compared. Specifically, impeded roots consumed O2 faster and O2 consumption was more likely to be O2-limited over a substantial proportion of the elongation zone, making these roots more susceptible to O2 deficit. Impeded roots used more O2 per unit growth (measured as either unit of elongation or unit of volumetric expansion) than unimpeded roots. Extension of impeded roots in situ was O2-limited at sub-atmospheric O2 levels (21% O2), while unimpeded roots were only limited below 11% O2. CONCLUSIONS: The shift in the zone of extension towards the apex in impeded roots coincided with greater vulnerability to hypoxia even after soil was removed. Roots still encased in impeded soil are likely to suffer from marked O2 deficits.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.