[Methylated analogues of spermine and spermidine as tools to investigate cellular functions of polyamines and the enzymes of their metabolism].

Biogenic amines spermine and spermidine are essential factors of cellular growth. Polyamine analogues are widely used to investigate and to regulate the enzymes of polyamine metabolism and functions of spermine and spermidine in vitro and in vivo. Recently, it was demonstrated that alpha-methylated derivatives of spermine and spermidine are capable to fulfill key cellular functions of polyamines, moreover in some cases of (R)- and (S)-isomers are actually different. Using these alpha-methylated spermine and spermidine analogues it turned possible to prevent the development of acute pancreatitis of SSAT-transgenic rats and to demostrate for the first time that polyamine oxidase, spermine oxidase and deoxyhypusine synthase have dormant stereospecificity. An original approach to regulate the stereospecificity of polyamine oxidase was suggested. It was also demonstrated that the depletion of the intracellular polyamine pool has both hypusine-related consequences and also the consequences unrelated to the posttranslational modification of eukaryotic initiation translation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogues for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.

Ectomycorrhizal fungi increase the vitality of Norway spruce seedlings under the pressure of Heterobasidion root rot in vitro but may increase susceptibility to foliar necrotrophs.

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.

Oxidative stress and inflammation in the pathogenesis of activated polyamine catabolism-induced acute pancreatitis.

The markers of oxidative stress and inflammation were studied in acute pancreatitis in transgenic rats exhibiting activated polyamine catabolism. In addition, the effect of bismethylspermine (Me(2)Spm) pretreatment, preventing pancreatitis in this model, on these mediators was investigated. Lipid peroxidation was increased at 6 and 24 h after induction of pancreatitis. These changes as well as the markedly decreased superoxide dismutase activity at 24 h were abolished by Me(2)Spm pretreatment. Glutathione level and catalase activity changed transiently, and the effect of Me(2)Spm was clear at 24 h. Serum inflammatory cytokine levels increased already at 4 h whereas NF-kappaB was distinctly activated only at 24 h. Me(2)Spm prevented the increase in TNF-alpha and IL-6 while it had no effect on NF-kappaB activation. These results show that typical inflammatory and, to a lesser degree, some oxidative stress mediators are involved and beneficially affected by the disease-ameliorating polyamine analogue in our pancreatitis model.

Mechanisms of polyamine catabolism-induced acute pancreatitis.

Acute pancreatitis is an autodigestive disease, in which the pancreatic tissue is damaged by the digestive enzymes produced by the acinar cells. Among the tissues in the mammalian body, pancreas has the highest concentration of the natural polyamine, spermidine. We have found that pancreas is very sensitive to acute decreases in the concentrations of the higher polyamines, spermidine and spermine. Activation of polyamine catabolism in transgenic rats overexpressing SSAT (spermidine/spermine-N(1)-acetyltransferase) in the pancreas leads to rapid depletion of these polyamines and to acute necrotizing pancreatitis. Replacement of the natural polyamines with methylated polyamine analogues before the induction of acute pancreatitis prevents the development of the disease. As premature trypsinogen activation is a common, early event leading to tissue injury in acute pancreatitis in human and in experimental animal models, we studied its role in polyamine catabolism-induced pancreatitis. Cathepsin B, a lysosomal hydrolase mediating trypsinogen activation, was activated just 2 h after induction of SSAT. Pre-treatment of the rats with bismethylspermine prevented pancreatic cathepsin B activation. Analysis of tissue ultrastructure by transmission electron microscopy revealed early dilatation of rough endoplasmic reticulum, probable disturbance of zymogen packaging, appearance of autophagosomes and later disruption of intracellular membranes and organelles. Based on these results, we suggest that rapid eradication of polyamines from cellular structures leads to premature zymogen activation and autodigestion of acinar cells.

Myb26: a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes.

Plant Myb proteins represent a group of transcription factors which have a DNA-binding domain similar to that found in the products of the animal myb proto-oncogenes. Members of the Myb family regulate the biosynthesis of phenylpropanoids, including anthocyanin and phlobaphene pigments, in several species. In this study, PCR with degenerate primers was used to analyse the presence of myb-like genes in pea (Pisum sativum L.). A fragment representing a flower bud-expressed gene, designated myb26, was recovered, and a full length cDNA was isolated from a pea flower bud cDNA library. The predicted protein is 217 amino acids long and its Myb-domain and carboxy terminal region show extensive homology to the snap-dragon proteins Myb305 and Myb340, both of which regulate phenylpropanoid biosynthesis. Expression of myb26 is flower-specific and parallels the expression of flavonoid biosynthetic genes, increasing as the flower bud matures and intensifies in colour. However, myb26 represents neither of the two known regulatory genes for anthocyanin production in pea, nor does it complement mutants of the myb-like anthocyanin regulatory gene an2 in petunia. Myb26 was expressed in E. coli as a fusion protein. It was shown that in vitro Myb26 recognizes the c-Myb and P-box-like binding sites representing cis-elements in the promoter regions of several phenylpropanoid biosynthetic genes. The results suggest that myb26 is a previously undefined gene involved in regulation of some aspect of phenylpropanoid production in pea.

Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize.

Anthocyanin production in higher plants is a function of the tissue considered and its developmental stage, and is modulated by environmental factors. In maize, the best characterized system, regulation of the pathway is achieved largely through the action of proteins with homology to the transcriptional factors encoded by myc and myb proto-oncogenes of animals; these homologues control the expression of structural genes and thus regulate the availability of anthocyanin biosynthetic enzymes. We have studied anthocyanin biosynthesis and its regulation in flowers of pea (Pisum sativum). Our results demonstrate a correlation between anthocyanin accumulation and steady-state mRNA levels for genes encoding chalcone synthase, flavanone 3 beta-hydroxylase, and dihydroflavonol 4-reductase in developing flowers. Patterns of expression for these biosynthetic genes in both a and a2 mutants confirm the regulatory roles of the two a loci. The reduced expression of all three biosynthetic genes in mutant lines suggests that genes acting both early and late in the anthocyanin biosynthetic pathway are controlled by a and a2. Particle bombardment of floral tissue demonstrates the ability of two maize R-like genes, Lc and R-S, but neither myb-like genes nor R-like genes from snapdragon or petunia, functionally to complement a and a2 mutations. We cannot distinguish whether a and a2 act coordinately or sequentially in anthocyanin regulation, but the epistatic action of maize R-like genes suggests that they mimic the action of a gene that normally functions downstream of both a and a2 in the regulatory cascade.

GRCD1, an AGL2-like MADS box gene, participates in the C function during stamen development in Gerbera hybrida.

Despite the differences in flower form, the underlying mechanism in determining the identity of floral organs is largely conserved among different angiosperms, but the details of how the functions of A, B, and C are specified varies greatly among plant species. Here, we report functional analysis of a Gerbera MADS box gene, GRCD1, which is orthologous to AGL2-like MADS box genes. Members of this group of genes are being reported in various species in growing numbers, but their functions remained largely unsettled. GRCD1 expression is detected in all four whorls, but the strongest signal is seen in the developing stamen and carpel. Downregulating GRCD1 expression by antisense transformation revealed that lack of GRCD1 caused homeotic changes in one whorl only: sterile staminodes, which normally develop in whorl 3 of marginal female florets, were changed into petals. This indicates that the GRCD1 gene product is active in determining stamen identity. Transgenic downregulation of GRCD1 causes a homeotic change similar to that in the downregulation of the Gerbera C function genes GAGA1 and GAGA2, but one that is limited to whorl 3. Downregulation of GRCD1 expression does not reduce expression of GAGA1 or GAGA2, or vice versa; and in yeast two-hybrid analysis, GRCD1 is able to interact with GAGA1 and GAGA2. We propose that a heterodimer between the GRCD1 and GAGA1/2 gene products is needed to fulfill the C function in whorl 3 in Gerbera.

Dual regulation by heat and nutrient stress of the yeast HSP150 gene encoding a secretory glycoprotein.

We have cloned and characterized the HSP150 gene of Saccharomyces cerevisiae, which encodes a glycoprotein (hsp150) that is secreted into the growth medium. Unexpectedly, the HSP150 gene was found to be regulated by heat shock and nitrogen starvation. Shifting the cells from 24 degrees C to 37 degrees C resulted in an abrupt increase in the steady-state level of the HSP150 mRNA, and de novo synthesized hsp150 protein. Returning the cells to 24 degrees C caused a rapid decrease in mRNA and protein synthesis to basal levels. The HSP150 5'-flanking region contains several heat shock element-like sequences (HSE). To study the function of these sequences, a strain bearing a disrupted copy of the HSP150 gene was transformed with plasmids in which the coding region of HSP150, or a HSP150-lacZ fusion gene, was preceded by 5' deletion derivatives of the HSP150 promoter. Site-directed mutagenesis of one HSE-like element, located between the TATA box and transcription initiation sites, abolished heat activation of transcription. In addition to heat shock, the HSP150 gene is regulated by the availability of nutrients in the growth medium. The HSP150 mRNA level was increased by nitrogen limitation at 24 degrees C, even when under the control of a HSP150 promoter region of 137 bp carrying the mutagenized HSE.

Gossypol activates pancreatic polyamine catabolism in normal rats and induces acute pancreatitis in transgenic rats over-expressing spermidine/spermine N1-acetyltransferase.

BACKGROUND: The male antifertility agent gossypol has been reported to induce spermidine/spermine N1-acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. METHODS: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma 2-amylase activity were determined after different doses of gossypol. RESULTS: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. CONCLUSIONS: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.