Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures

Endophytes are found in meristematic bud tissues of Scots pine ( Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial ( Methylobacterium extorquens , Pseudomonas synxantha ) and one fungal endophyte ( Rhodotorula minuta ) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.     

Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting (14)C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.     

Localization and functional analysis of PepI, the immunity peptide of Pep5-producing Staphylococcus epidermidis strain 5

Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.     

Bone and joint neuropathy in rats with type-2 diabetes

We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia.     

The actin cytoskeleton in normal and pathological cell motility

Cell motility is crucial for tissue formation and for development of organisms. Later on cell migration remains essential throughout the lifetime of the organism for wound healing and immune responses. The actin cytoskeleton is the cellular engine that drives cell motility downstream of a complex signal transduction cascade. The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. The multitude of proteins and regulatory mechanisms partaking in this system makes it vulnerable to mutations and alterations in expression levels that ultimately may cause diseases. The most familiar one is cancer that in later stages is characterized by active aberrant cell migration. Indeed tumor invasion and metastasis are increasingly being associated with deregulation of the actin system.     

The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs

In order to determine the effect of pectin on fermentation parameters in the faeces and caecal digesta of weaned pigs 18 castrated male crossbred pigs with an average body weight of 8 kg were fitted with T-cannulas at the caecum. The animals were randomly distributed into three groups and fed with diets supplemented with 0, 5 and 10% pectin. Faeces were collected over a period of 3 days. Thereafter the diets were withdrawn for 24 h followed by ad libitum feeding to enhance the feed intake. Caecal chyme was collected 0, 8 and 24 h postprandial. In the faeces the addition of 5% pectin to the diet lowered the content of dry matter and lactic acid. The pH and the digestibility of pectins, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. When the diets were withdrawn and fed ad libitum 24 h later, a decline of the pH and an increased concentration of lactate in the caecal chyme could be observed in all groups up to 8 h after feeding. With an interval of 8 to 24 h after feeding, a further decline in pH and a rise of lactate only occurred when the diet was not supplemented with pectin. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine.     

Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: interactions of thioredoxin with elongation factor 1alpha and yeast alcohol dehydrogenase

Thioredoxins (Trx) are ubiquitous dicysteine proteins capable of modulating enzymes and other cellular targets through specific disulfide-dithiol redox changes. They are unique in that a large number of very diverse metabolic systems are addressed and redox-regulated in bacteria, animal, and plant cells, but the finite number of thioredoxin interaction partners is still unknown. Two-hybrid methodology should provide a rational way to establish thioredoxin functions in a given organism. We report a search for physiological target proteins of thioredoxin1 in the social amoeba Dictyostelium discoideum , which possesses three developmentally regulated thioredoxin genes, all of which lack functional characterisation. A two-hybrid approach identified at least seven bona fide thioredoxin partners, including oxidoreductases, proteins of the ribosomal translation apparatus, and the cytoskeletal protein filopodin. With the exception of ribonucleotide reductase, none of these systems had previously been linked to specific redox modulation. Molecular interactions in two of the new thioredoxin/target protein couples were verified by biochemical studies: (1) thioredoxin1 and the abundant elongation factor 1alpha from D. discoideum form the mixed heterodisulfide characteristic of the thioredoxin mechanism of action; and (2) reduced thioredoxin, but not glutathione, strongly inhibits yeast alcohol dehydrogenase catalysis of ethanol oxidation.     

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase

Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-vellosimine (or gardneral). The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina. Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli. The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 microM. The amino acid sequence of vinorine synthase has highest level of identity (28-31%) to that of Papaver salutaridinol acetyltransferase, Fragaria alcohol acyltransferase, and Catharanthus deacetylvindoline acetyltransferase involved in morphine, flavor, and vindoline biosynthesis, respectively. Vinorine synthase is a novel member of the BAHD superfamily of acyltransferases. Site-directed mutagenesis of 13 amino acid residues provided clear evidence that both, His160 and Asp164 of the consensus sequence HxxxD belong to the catalytic center. The mutations also showed that an amino acid triad is not characteristic of vinorine synthase. The experiments demonstrated the importance of the conserved motif SxL/I/VD near the N-terminus and the consensus sequence DFGWG near the C-terminal.