Sensitization to the hyperthermic and catecholamine-releasing effects of morphine

Rats were given alternating injections (one per day) of morphine (5 mg/kg) and saline, each substance paired with a distinct set of environmental cues. Over the course of nine injections of the drug, the hyperthermic response to morphine gradually increased. On the tenth such exposure to morphine, half of the rats were injected in the presence of cues previously paired with the drug, and half were injected in the presence of saline cues. In the former (drug-cue) group, hyperthermia, and plasma norepinephrine and epinephrine levels were significantly greater than in rats receiving morphine for the first time. The latter (saline-cue) group showed an intermediate thermic and catecholamine response, not statistically different from drug-cue or control animals. Under these conditions, the enhanced hyperthermic response to morphine (which has been variously described as sensitization or tolerance), was found to be accompanied by a similar increase in elevation of plasma catecholamines.     

1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5

1H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8-nporphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate at the crystallographic 8-methyl position was a significant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond at the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.     

Endo-β-1,4-glucanases impact plant cell wall development by influencing cellulose crystallization

Cell walls are vital to the normal growth and development of plants as they protect the protoplast and provide rigidity to the stem. Here, two poplar and Arabidopsis orthologous endoglucanases, which have been proposed to play a role in secondary cell wall development, were examined. The class B endoglucanases, Pt GH9B5 and At GH9B5, are secreted enzymes that have a predicted glycosylphosphatidylinositol anchor, while the class C endoglucanases, Pt GH9C2 and At GH9C2, are also predicted to be secreted but instead contain a carbohydrate-binding module.The poplar endoglucanases were expressed in Arabidopsis using both a 35 S promoter and the Arabidopsis secondary cell wall-specific Ces A8 promoter. Additionally, Arabidopsis t-DNA insertion lines and an RNAiconstruct was created to downregulate At GH9C2 in Arabidopsis. All of the plant lines were examined for changes in cell morphology and patterning, growth and development, cell wall crystallinity, micro fibril angle, and proportion of cell wall carbohydrates. Misregulation of Pt GH9B5/At GH9B5 resulted in changes in xylose content, while misregulation of Pt GH9C2/At GH9C2 resulted in changes in crystallinity, which was inversely correlated with changes in plant height and rosette diameter. Together, these results suggest that these endoglucanases affect secondary cell wall development by contributing to the cell wall crystallization process.     

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.     

Amphioxus tails: source and fate of larval fin rays and the metamorphic transition from an ectodermal to a predominantly mesodermal tail

It was previously discovered that tail fin rays of larval amphioxus are long ciliary rootlets in posterior epidermal cells. This work describes the heretofore unknown origin and fate of these organelles in the Florida amphioxus (Branchiostoma floridae). In late embryos, epidermal cells at the posterior end of the body increase in height, thus producing a tail fin. One ciliary rootlet in each cell elongates and also rotates through about 90°, soon becoming oriented parallel to the long axis of the cell and running continuously from the apical to the basal plasma membrane. During the subsequent growth of the larval tail, the rootlets and epidermal cells housing them reach lengths up to 120 μm. At metamorphosis, the rootlets become vacuolated and rapidly decrease in length along with the height of the tail epidermis. Contemporaneously, abundant extracellular dermal matrix accumulates in the sagittal plane of the body to produce a predominantly dermal tail fin. Throughout postmetamorphic life, the posterior epidermal cells, now without ciliary rootlets, thinly cover a largely dermal tail flange. Thus, the specialized morphology of the amphioxus tail fin is generated by two different cellular mechanisms, involving different cell populations (ectodermal and mesodermal), at different life‐history stages.     

Unidirectional Movement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in Mucilage Extrusion,Adherence, and Ray Formation

CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.The epidermal cells of Arabidopsis (Arabidopsis thaliana) seed coats produce two distinct secondary cell walls: pectin-rich mucilage and cellulose-rich columellae (Western et al., 2000). When seeds are hydrated, mucilage expands rapidly, rupturing the outer tangential cell wall and forming a mucilage capsule that surrounds the seed. Seed coat mucilage is composed primarily of rhamnogalacturonan I (RG I) and also contains homogalacturonan (HG), hemicelluloses (such as xylans and glucomannans), and cellulose (for review, see Haughn and Western, 2012). Extruded mucilage consists of an outer, nonadherent fraction and an inner, adherent fraction (Western et al., 2000, 2001; Macquet et al., 2007a). The adherent and nonadherent mucilage layers differ in the amount of methylesterified HG (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013), galactans (Dean et al., 2007; Macquet et al., 2007b), arabinans (Arsovski et al., 2009), mannans (Yu et al., 2014), and cellulose (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011), all of which influence the physical properties of the layers.Adherent mucilage has a distinct structure, which can be examined using cell wall dyes and antibodies. When treated with cellulose-specific dyes, densely stained rays extend from the top of each columella to the outer edge of the adherent layer, many cell lengths above the seed surface (Mendu et al., 2011; Sullivan et al., 2011). Cytological evidence indicates that cellulose, pectins, and mannans are components of the ray (Haughn and Western, 2012; Griffiths et al., 2014; North et al., 2014; Yu et al., 2014), although the exact manner in which they are assembled is unknown.Cellulose is abundant in mucilage rays and mediates adherence. Loss-of-function mutations in CELLULOSE SYNTHASE5 (CESA5) result in reduced cellulose levels and increased detachment of mucilage from the seed (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014). How a reduction in cellulose results in a loss of adherence is still unknown, but it likely involves interaction with other mucilage components such as pectin and arabinogalactan proteins (Griffiths et al., 2014). Since cesa5 mutants still have some cellulose in the rays of the adherent mucilage halo (Mendu et al., 2011; Sullivan et al., 2011), additional cellulose synthases must be involved in mucilage cellulose biosynthesis.The Arabidopsis genome encodes 10 different CESAs (Delmer, 1999; Richmond and Somerville, 2000). Multiple lines of evidence suggest that three different CESAs are required to form one active cellulose synthase complex (CSC; for review, see Somerville, 2006). CSCs are membrane-bound protein complexes that synthesize cellulose microfibrils in the apoplast (for review, see Somerville, 2006; Endler and Persson, 2011; Lei et al., 2012). CESA1, CESA3, and CESA6 are considered the core components of the primary wall CSC (Desprez et al., 2007; Persson et al., 2007). CESA2, CESA5, and CESA9 are partially redundant to CESA6 in primary wall biosynthesis, and genetic evidence suggests that each of these CESA polypeptides can form a functional CSC with CESA3 and CESA1 (Desprez et al., 2007; Persson et al., 2007). CESA10 is expressed in young plants, stems, floral tissue, and the base of rosette leaves (Beeckman et al., 2002; Doblin et al., 2002), but its function in cellulose biosynthesis is unclear. Other cesa mutant lines have been examined for altered mucilage phenotypes (cesa1, radially swollen1 [Burn et al., 2002; Sullivan et al., 2011], cesa2, cesa6, and cesa9 [Mendu et al., 2011]; CESA3, je5 [Sullivan et al., 2011] and cesa10-1 [Sullivan et al., 2011]); to date, only CESA5 has been shown to be required for cellulose biosynthesis during mucilage deposition.Two mutant alleles of CESA3, isoxaben resistant1-1 (ixr1-1) and ixr1-2, were isolated in a screen for resistance to the herbicide isoxaben (Scheible et al., 2001). Isoxaben inhibits the incorporation of Glc into the emerging cellulose polymer and is considered a potent and specific inhibitor of cellulose biosynthesis (Heim et al., 1990). Homozygous ixr1-1 and ixr1-2 lines show increased resistance to the herbicide, and the mutations causing this resistance were mapped to the genomic locus of CESA3 (Heim et al., 1990; Scheible et al., 2001). The ixr1-1 and ixr1-2 mutations cause amino acid substitutions near the C terminus of the CESA3 protein. ixr1-1 causes a Gly-to-Asn substitution (G998A) located in a transmembrane domain, while ixr1-2 contains a Thr-to-Ile substitution (T942I) in an apoplastic region of the protein between two transmembrane domains (Scheible et al., 2001). Recently, the ixr1-2 allele was shown to affect the velocity of CSCs in the plasma membrane, which consequently modifies cellulose crystallinity in the cell wall (Harris et al., 2012). It is not exactly clear how the ixr1-1 mutation affects cellulose biosynthesis. The effects of either of these mutations on seed coat mucilage have not been investigated.Since mucilage is composed primarily of pectins with smaller amounts of cellulose, seed coat epidermal cells represent an excellent system to study cellulose biosynthesis and interactions between cellulose and other wall components in muro. In this study, we investigated how cellulose is synthesized and deposited in seed coat epidermal cells. We show that at least three different CESA proteins are highly expressed in the seed coat epidermis during mucilage biosynthesis. These CESAs are oriented and move in a linear fashion around the cytoplasmic column of each cell in an identical pattern to cortical microtubules. In addition, we provide evidence that the adherent mucilage has a helical structure that expands and unwinds during extrusion to form the mucilage ray. We propose that during seed coat epidermal cell development, the biosynthesis of cellulose predetermines the structure of rays in the adherent mucilage layer.