Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Plant mRNA 3′-end formation

Our understanding of how the 3 ends of mRNAs are formed in plants is rudimentary compared to what we know about this process in other eukaryotes. The salient features of plant pre-mRNAs that signal cleavage and polyadenylation remain obscure, and the biochemical mechanism is as yet wholly uncharacterised. Nevertheless, despite the lack of universally conserved cis-acting motifs, a common underlying architecture is emerging from functional analyses of plant poly(A) signals, allowing meaningful comparison with components of poly(A) signals in other eukaryotes. A plant poly(A) signal consists of one or more near-upstream elements (NUE), each directing processing at a poly(A) site a short distance downstream of it, and an extensive far-upstream element (FUE) that enhances processing efficiency at all sites. By analogy with other systems, a model for a plant 3-end processing complex can be proposed. Plant poly(A) polymerases have been isolated and partially characterised. These, together with hints that some processing factors are conserved in different organisms, opens promising avenues toward initial characterisation of the trans-acting factors involved in 3-end formation of mRNAs in higher plants.     

Thermoluminescence from the photosynthetic apparatus

One of the fundamental discoveries of W. Arnold was the detection of thermally stimulated light emission from preilluminated photosynthetic material (Arnold and Sherwood (1957) Proc Natl Acad Sci USA 43: 105–114). This phenomenon, called thermoluminescence (TL), is characteristic of a wide range of materials (semiconductors, minerals, inorganic and organic crystals, and complex biological systems such as the photosynthetic apparatus) which share the common ability of storing radiant energy in thermally stabilized trap states.The original discovery of TL in dried chloroplasts later proved to be a phenomenon common to all photosynthetic organisms: photosynthetic bacteria, cyanobacteria, algae and higher plants. Following the pioneering work of Arnold, considerable effort has been devoted to identification and characterization of photosynthetic TL components. This work has firmly established the participation of various redox states of the water-oxidizing complex and the quinone electron acceptors of Photosystem II in the generation of photosynthetic glow curves. Since TL characteristics are very sensitive to subtle changes in redox properties of the involved electron transport components, the TL method has become a powerful tool in probing a wide range of PS II redox reactions. In this paper, we will review the impact of Arnold's work in initiating and promoting TL studies in photosynthesis and will cover the most important developments of this field of research until the present day.Abbreviations Chl chlorophyll - DL delayed luminescence - PS photosystem - TL thermoluminescence     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of -d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).