Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.     

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.     

Plasma and liver selenium levels in the rat during supplementation with 0.5, 2, 6, and 15 ppm selenium in drinking water

Plasma and liver selenium of Wistar rats were determined after 1, 3, and 6 mo supplementation with 0.5, 2, 6, or 15 ppm selenium as sodium selenite in drinking water. Plasma selenium was not different from control values at additional intake of 0.5 ppm but increased above usual levels at higher intakes. A highly significant correlation was observed between the total quantity of selenium ingested and plasma selenium after 1 mo treatment (r=0.99,p<0.01), but was less pronounced after 3 and 6 mo (0.94,p<0.05, and 0.78,p<0.05, respectively). The decrease in plasma selenium with time of treatment was more pronounced at higher intakes. There was also a highly significant correlation between total selenium intake and liver selenium concentration (r=0.99,p<0.01) after 1 mo of treatment, but this time liver selenium did not change with time, and the correlation remained highly significant throughout the investigation. Liver selenium therefore appears as a more sensitive and more representative measure of selenium intake than plasma selenium. Most supplements did not affect body weight and survival of animals, except when the diet was supplemented with 15 ppm for 6 mo; however, alterations in biochemical parameters concerning lipid status and hepatic function were observed at levels above 2.0 ppm.     

Influence of ovule perforation,plant growth regulators,andl-glutamine on in vitro growth of immature peach embryos

Summary Ovule perforation technique and media components (plant growth regulators andl-glutamine) were tested on in vitro growth of immature (<3 mm) embryos of “Springcrest” and “Earligrande” peaches. Ovule perforation was 2 to 4 times more effective in promoting embryo growth than leaving ovules intact.l-Glutamine (400 mg·liter⁻¹) promoted an increase in growth but could not be used with indole-acetic acid plus kinetin because an antagonistic effect on embryo growth occurred. The use of these exogenous plant growth regulators did not increase embryo growth over in vivo growth.     

Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day⁻¹ resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m⁻³ day⁻¹. However, higher PHB productivities (~127 g PHB m⁻³ day⁻¹) were achieved using nitrogen excess at a D = 0.2 day⁻¹. Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.     

Canopy tree mortality depends on the proportion of crown exposed to sunlight,but this effect varies with species' wood density

Understanding what drives changes in tree mortality as well as the covariates influencing trees' response is a research priority to predict forest responses to global change. Here, we combined drone photogrammetry and ground-based data to assess the influence of crown exposure to light (relative to total crown area), growth deviations (relative to conspecifics), tree size, and species' wood density (as a surrogate for light-demanding and shade-tolerant life-history strategies) on the mortality of 984 canopy trees in an Amazon terra firme forest. Trees with lower wood density were less prone to die when their proportion of crown was more exposed to sunlight, but this relationship with relative crown exposure weakened and slightly reversed as wood density increased. Trees growing less than their species average had higher mortality, especially when the species' wood density decreased. The role of wood density in determining the survival of canopy trees under varying light conditions indicates differential responses of light-demanding versus shade-tolerant species. Our results highlight the importance of accounting for life-history strategies, via plant functional types, in vegetation dynamic models aiming to predict forest demography under a rapidly changing climate. Abstract in Spanish is available with online material.