Recent advances in chemiluminescence-based immunoassays for steroid hormones

Several formats of solid-phase separation techniques for the measurement of steroids and urinary steroid conjugates using chemiluminescence as an end point are described. These formats include: (1) immunoadsorption of second antibodies directed against the first antibody on solid support; (2) specific immunoadsorbents consisting of primary antibodies covalently coupled to polymer beads; (3) second antibody coupled to a polymer containing magnetic particles. In these assays a steroid-chemiluminescent marker conjugate serves as the labelled ligand, and highly specific homologous monoclonal antibodies are used to provide optimal specificity and rigorous standardization. These techniques enabled the direct measurement of steroid glucuronides in diluted urine and of steroids in plasma.     

Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.     

Specific Inhibition of Lignification in Bryonia dioica: Effects on Thigmomorphogenesis

Rubbing-induced lignification in Bryonia dioica internodes is significantly impaired by N(o-hydroxyphenyl) and N(o-aminophenyl) sulfinamoyl tertiobutyl acetate, specific inhibitors of cinnamyl alcohol dehydrogenase, an enzyme which is strictly associated with lignin monomer synthesis. Along with the reduction of lignification, these inhibitors counteract the inhibition of elongation due to rubbing. These results indicate that lignification participates in the thigmomorphogenetic growth response of Bryonia dioica internodes. In a general way, the data point to the causal role of lignification in the limitation of plant growth.     

Site-specific integration and excision of pMEA100 in Nocardia mediterranei

Summary Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Biphasic fluence-response curves for phytochrome-mediated kalanchoë seed germination : sensitization by gibberellic Acid

The fluence-response curves for the effect of two red pulses separated by 24 hours on the germination of Kalanchoe blossfeldiana Poelln. cv Vesuv seeds, incubated on gibberellic acid (GA₃) are biphasic for suboptimal concentrations. The response in the low fluence range corresponds with a classical red/far-red reversible phytochrome mediated reaction. GA₃ induces an additional response in the very low fluence range, which is also phytochrome mediated. The sensitivity to phytochrome-far-red absorbing form (Pfr), however, is increased about 20,000-fold, so that even far-red fluences become saturating. Both in the very low and low fluence response range, the maximal responses induced by saturating fluences are modulated by the GA₃ concentration. GA₃ having no direct influence on the phytochrome phototransformations, alters the Pfr requirement and determines the responding seed population fraction in the very low and low fluence range. The effet of GA₃ appears to be on the transduction chain of the phytochrome signal.     

Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers

Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not.     

Relative biological effectiveness (RBE) of neutrons produced by 50 MeV deuterons and by 34, 45, 65 and 75 MeV protons

RBE of p(34) + Be, p(45) + Be, p(65), + Be, p(75) + Be and d(50) + Be neutron beams produced at the cyclotron "Cyclone" of Louvain-la-Neuve were measured. The biological criterion was the regeneration of the crypts of the intestinal mucosa (50 regenerated crypts per circumference) after abdominal irradiation in mice. Taking the p(65) + Be neutrons as reference, RBE values were found equal to 1.12, 1.07, 1.00 (Ref.), 0.96 and 1.02 respectively. These results are consistent with those published for cell lethality in vitro. However, the RBE variation is smaller than this previously obtained in the laboratory for growth inhibition in Vicia faba.     

Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L.) Candargy

Summary The zymogram phenotypes of glucose-phosphate isomerase (GPI), alcohol dehydrogenase-1 (ADH-1), glutamate oxaloacetate transaminase (GOT), superoxide dismutase (SOD), lipoxygenase (LPX), esterase (EST) and the banding patterns of gliadin and glutenin seed storage proteins were determined for Triticum aestivum cv. Chinese Spring (CS), Dasypyrum villosum, the octoploid amphiploid T. aestivum cv. Chinese Spring D. villosum (CS × v) (2n=8x=56; AABBDDVV), and for five CS-D. villosum disomic addition lines. The genes Gpi-V1, Adh-V1, Got-V2, and Sod-V2 coding for GPI-1, ADH-1, GOT-2, and SOD-2 isozymes were located in D. villosum on chromosome 1V, 4V, 6V, and 7V, respectively. Genes coding for gliadin- and glutenin-like subunits are located in D. villosum chromosomes 1V. There are no direct evidence for chromosomal location of genes coding for GOT-3, EST-1 and LPX-2 isozymes. The linkage between genes coding for glutenin-like proteins and GPI-1 isozymes in chromosome 1V is evidence of homoeology between chromosome 1V and the chromosomes of homoeologous group 1 in wheat.Research supported by the National Research Council (Italy) and National Science Foundation (USA). International cooperative project, Grant No. 85.01504.06 (CNR)