Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)

Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B₅) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and 0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter) before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of nodulelike structures on basal medium (B₅) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios.     

Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata

Endogenous indole-3-acetic acid, abscisic acid and cytokinins (zeatin, zeatin riboside, N-isopentenyladenine and N-isopentenyladenosine) were evaluated in initial explants (leaves) of in vitro propagated plants of alfalfa ( Medicago falcata L.) lines varying in embryogenic capacity and during the somatic embryogenesis process. Fast embryo-genic induction was correlated with high IAA and low ABA levels in the initial explants. No significant differences were observed in the cytokinin contents. Our results suggest that a certain hormone balance is necessary to allow the expression of the embryogenic potential. The consistent stages of the direct somatic embryogenesis are also characterized by changes in hormonal levels.     

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches

The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.     

Proteomic Biomarkers Associated with Streptococcus agalactiae Invasive Genogroups

Group B streptococcus (GBS, Streptococcus agalactiae) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (i.e. ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.     

Direct somatic embryogenesis and plant regeneration of carnation (Dianthus caryophyllus L.)

Conditions for efficient direct somatic embryogenesis and plant regeneration of leaf explants from carnation cultivars Lena (SIM group) and Bulgarian spray cultivars Nasslada, Yanita, Regina and Line 84 were established. Murashige and Skoog (MS) liquid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-benzylaminopurine was used for direct induction of embryoids without an additional callus phase. The first globular structures were observed after 20 days of cultivation. Their further development to the torpedo stage was correlated with the presence of polyethylene glycol (PEG 6000). Somatic embryo maturation was promoted by casein hydrolysate (1000 mg/l) in MS liquid media. The percentage conversion of embryos and polyembryos to whole plants varied between 10 and 75% among studied cultivars. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in a greenhouse. Received: 29 November 1996 / Revision received: 28 April 1997 / Accepted: 28 May 1998     

Immunolocalization of hypochlorite-induced, catalase-bound free radical formation in mouse hepatocytes

The establishment of oxidants as mediators of signal transduction has renewed the interest of investigators in oxidant production and metabolism. In particular, H(2)O(2) has been demonstrated to play pivotal roles in mediating cell differentiation, proliferation, and death. Intracellular concentrations of H(2)O(2) are modulated by its rate of production and its rate of decomposition by catalase and peroxidases. In inflammation and infection, some of the H(2)O(2) is converted to hypochlorous acid, a key mediator of the host immune response against pathogens. In vivo HOCl production is mediated by myeloperoxidase, which uses excess H(2)O(2) to oxidize Cl(-). Mashino and Fridovich (Biochim. Biophys. Acta 956:63-69; 1988) observed that a high excess of HOCl over catalase inactivated the enzyme by mechanisms that remain unclear. The potential relevance of this as an alternative mechanism for catalase activity control and its potential impact on H(2)O(2)-mediated signaling and HOCl production compelled us to explore in depth the HOCl-mediated catalase inactivation pathways. Here, we demonstrate that HOCl induces formation of catalase protein radicals and carbonyls, which are temporally correlated with catalase aggregation. Hypochlorite-induced catalase aggregation and free radical formation that paralleled the enzyme loss of function in vitro were also detected in mouse hepatocytes treated with the oxidant. Interestingly, the novel immuno-spin-trapping technique was applied to image radical production in the cells. Indeed, in HOCl-treated hepatocytes, catalase and protein-DMPO nitrone adducts were colocalized in the cells' peroxisomes. In contrast, when hepatocytes from catalase-knockout mice were treated with hypochlorous acid, there was extensive production of free radicals in the plasma membrane. Because free radicals are short-lived species with fundamental roles in biology, the possibility of their detection and localization to cell compartments is expected to open new and stimulating research venues in the interface of chemistry, biology, and medicine.     

Chlorsulfuron resistant transgenic tobacco as a tool for broomrape control

Broomrape (Orobanche ramosa L.) is the most important parasitic plant that infests tobacco (Nicotiana tabacum L.). Chemical treatment of the soil is not effective and crop rotation is not acceptable to solve this problem because of the long viability period of Orobanche seeds in the soil. Application of systemic herbicides in the field with herbicide resistant tobacco could be a successful tool for broomrape control. Several tobacco cultivars were transformed with a mutant ahas3R gene for resistance to the herbicide chlorsulfuron (Glean^®, DuPont). Transformed plants were selfed and the segregation of resistance was followed in the next generation. The efficiency of the herbicide was demonstrated in greenhouse and field trials. An Orobanche/tobacco growth system was used in order to prove the lethal effect of the herbicide to the attached broomrape plants.     

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

Design of Carbon Nanotube‐Based Gas‐Diffusion Cathode for O2 Reduction by Multicopper Oxidases

Multicopper oxidases, such as laccase or bilirubin oxidase, are known to reduce molecular oxygen at very high redox potentials, which makes them attractive biocatalysts for enzymatic cathodes in biological fuel cells. By designing an enzymatic gas‐diffusion electrode, molecular oxygen can be supplied through the gaseous phase, avoiding solubility and diffusion limitations typically associated with liquid electrolytes. In doing so, the current density of enzymatic cathodes can theoretically be enhanced. This publication presents a material study of carbon/Teflon composites that aim to optimize the functionality of the gas‐diffusion and catalytic layers for application in enzymatic systems. The modification of the catalytic layer with multiwalled carbon nanotubes, for example, creates the basis for stronger π–π stacking interactions through tethered enzymatic linkers, such as pyrenes or perylene derivates. Cyclic voltammograms show the effective direct electron contact of laccase with carbon nanotube‐modified electrodes via tethered crosslinking molecules as a model system. The polarization behavior of laccase‐modified gas‐diffusion electrodes reveals open‐circuit potentials of +550 mV (versus Ag/AgCl) and current densities approaching 0.5 mA cm² (at zero potential) in air‐breathing mode.