Temporary immersion bioreactors (TIB) provide a versatile,cost-effective and reproducible in vitro analysis of the response of pineapple shoots to salinity and drought

Effects of salinity (NaCl) and the carbon source mannitol (0–200 mM) on micropropagation of pineapple cv. MD2 were analyzed in temporary immersion bioreactors (TIBs). Shoot multiplication rate, shoot cluster fresh weight and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. The content of soluble phenolics in the culture medium was also evaluated. NaCl or mannitol above concentrations of 50 mM decreased pineapple shoot multiplication and fresh weight significantly. Two hundred mM NaCl decreased multiplication rate by 71.5% and cluster fresh weight by 40.0%. NaCl increased 2.4 times the levels of other aldehydes; 1.4 times the soluble phenolics in shoots; and 1.4 times the phenolics excreted to the culture medium. On the other hand, mannitol decreased the multiplication rate and cluster fresh weight by about 60%. Mannitol increased the contents of chlorophyll b 1.4 times and soluble phenolics 2.1 times. Results indicated that pineapple cv. MD2 is more sensitive to NaCl than to mannitol. Multiplication rates indicate that a 50% reduction was obtained with 37.4 mM NaCl and 66.5 mM mannitol. These concentrations can be used to stress shoots during micropropagation in TIBs and screen for/detect somaclonal variants with an increased salinity or drought tolerance.     

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.     

Angiotensin-(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1–7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from ³H- l -tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1–7) showed a significant decrease in TH specific activity. Ang-(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT₂ receptor antagonist and not by an AT₁ or Mas receptor antagonist, suggesting the involvement of AT₂ receptors. The proteasome inhibitor MG132 blocked the Ang-(1–7)-mediated TH reduction. In addition, Ang-(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang-(1–7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1–7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.     

p8/nupr1 regulates DNA‐repair activity after double‐strand gamma irradiation‐induced DNA damage

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)‐associated protein, which in turn binds the DNA‐damage‐associated 53BP1 protein to facilitate DNA repair following DNA γ‐irradiation. Contrary to the HAT‐associated activity, MSL1‐dependent DNA‐repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1‐dependent DNA‐repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after γ‐irradiation, it is eventually submitted to sustained down‐regulation, presumably to allow development of MSL1‐associated DNA‐repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1‐dependent HAT activity to the vicinity of damaged DNA. MSL1‐dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery. J. Cell. Physiol. 221: 594–602, 2009. © 2009 Wiley‐Liss, Inc.     

Calcium occlusion in plasma membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.     

Vesicle-associated membrane protein-2 (VAMP2) mediates cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ~50% and impaired cAMP-stimulated exocytosis by ~50%, as monitored with FM1-43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ~50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.