Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.     

Recruitment,mortality and growth of mangrove (<Emphasis Type="Italic">Rhizophora</Emphasis> sp.) seedlings in Ulugan Bay,Palawan, Philippines

Propagule dispersal, establishment and recruitment to the sapling stage are critical steps in the life cycle of mangroves. Specific (i.e., per capita) rates of recruitment and mortality, and the growth rates of Rhizophora seedlings in three mangrove stands in Ulugan Bay (Palawan, Philippines) were estimated between March 1999 and February 2001. Recruitment and mortality were variable in space and time, with mortality exceeding rates of recruitment at all sites. The specific rates of seedling recruitment and mortality were higher in Buenavista (0.66 year^–1 and –1.67 year^–1) than in Umalagan (0.05 year^–1 and –0.33 year^–1) and Oyster Bay (0.13 year^–1 and –0.24 year^–1). The annual rate of production of internodes by the main stem was similar at the three sites (5.4–5.5 internodes year^–1), but the annual rate of elongation of the main stem was higher in Buenavista (10.6 cm year^–1) than in Oyster Bay (7.6 cm year^–1) and Umalagan (5.6 cm year^–1).     

Rose Bengal located within liposome do not affect the activity of inside-out oriented Na,K-ATPase

DPPC:DPPE-proteoliposomes (in which the enzyme is inside-out oriented) and DLOPC:DLOPE-proteoliposomes (in which the enzyme is only 40% inside-out oriented) is an excellent model for studying the selective effect of the reactive oxygen species, produced by the photo-activation of Rose Bengal. Both proteoliposomes used, when submitted to photo-irradiation with laser using 1200 mJ/cm2 energy dose, in the absence of the Rose Bengal, did not shown any effect in the ATPase activity and in the integrity of its systems. Also, no effect was observed using 50 microM of Rose Bengal encapsulated in the interior of the DPPC:DPPE-proteoliposome system. But, when we use 50 microM of Rose Bengal, present only in the extravesicular environment, and photo-irradiation with a laser dose of 200 mJ/cm2, it results in the loss of 40-50% of the ATPase activity, with damage of the DPPC:DPPE-proteoliposome integrity. Using a dose of 400 mJ/cm2 the ATPase activity was totality lost. Consequently, these effects could be correlated with direct damage in the peptide structure. The photo-irradiation of the system constituted by DLOPC:DLOPE-proteoliposome in the presence of Rose Bengal, encapsulated only in the interior compartment or in the extra-liposomal environments, revealed a gradual decrease of the ATPase activity, maintaining it at 30% after a dose of 1200 mJ/cm2 and losing total ATPase activity at 800 mJ/cm2, respectively, with the loss of integrity of this vesicular system in both conditions studied. The generated singlet oxygen could attack the double linkage present in the fatty acid structure of the lipid instead of the amino acid in the protein structure and, in a second step, result in an indirect inactivation of the enzyme activity. In summary, these results indicated that singlet oxygen species produced by photo-oxidation of Rose Bengal using laser light could act in protein and lipid structure depending on its proportion or distribution.     

Role of PGI2 and effects of ACE inhibition on the bradykinin potentiation by angiotensin-(1-7) in resistance vessels of SHR

The present study determined the participation of PGI2 in the angiotensin-(1-7) [Ang-(1-7)]/bradykinin (BK) interaction, in the presence and absence of Angiotensin Converting Enzyme (ACE) inhibition, trying to correlate it with tissue levels of both peptides. The isolated mesenteric arteriolar bed of Spontaneously Hypertensive Rats (SHR) was perfused with Krebs or Krebs plus enalaprilat (10 nM), and drugs were injected alone or in association. BK (10 ng)-induced relaxation was potentiated by Ang-(1-7) (2.2 microg) in the presence or absence of enalaprilat. Ang-(1-7) receptor blockade [A-779 (4.8 microg)] did not interfere with the BK effect in preparations perfused with normal Krebs, but reversed the increased BK relaxation observed after ACE inhibition. PGI2 release by mesenteric vessels was not altered by BK or Ang-(1-7) alone, but was increased when both peptides were injected in association, in the absence or in the presence of enalaprilat. ACE inhibition caused a 2-fold increase in the BK tissue levels, and a significant decrease in the Ang-(1-7) values. We conclude that endogenous Ang-(1-7) has an important contribution to the effect of ACE inhibitors participating in the enhancement of BK response. The mechanism of Ang-(1-7) potentiating effect probably involves an increased production of PGI2. Our results suggest that a different enzymatic pathway (non-related to ACE) is involved in the local Ang-(1-7) metabolism.     

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.     

Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes

To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate-limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or -occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume.     

Contribution of N from green harvest residues for sugarcane nutrition in Brazil

Brazil is recognized as a prominent renewable energy producer due to the production of ethanol from sugarcane. However, in order for this source of energy to be considered truly sustainable, conservation management practices, such as harvesting the cane green (without burning) and retaining the trash in the field, need to be adopted. This management practice affects mostly the nitrogen (N) cycle through the effect of trash on immobilization–mineralization of N by soil microorganisms. The aim of the experiments reported here was to evaluate N recovery from trash (trash‐N) by sugarcane during three ratoon crop seasons: 2007, 2008 and 2009. Two field experiments were carried out, one in Jaboticabal and the other in Pradopolis, in the state of Sao Paulo, Brazil. The experiments were set up in a randomized block design with four replications. Within each plot, microplots were installed where the original trash was replaced by trash labelled with ¹⁵N, and maintained up to the fourth crop cycle. Trash‐N recovery was higher in the Jaboticabal site, the most productive one, than in the Pradópolis site. The average trash‐N recovery across the two sites after three crop cycles was 7.6 kg ha^?1 (or 16.2% of the initial N content in trash), with the remaining trash‐N being incorporated into soil organic matter reserves. While these results indicate that the value of trash for sugarcane nutrition is limited in the short term, maintaining trash on the field will serve as a long‐term source of N and C for the soil.