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.     

Regulation and functional significance of phospholipase D in myocardium

There is now clear evidence that receptor-dependent phospholipase D is present in myocardium. This novel signal transduction pathway provides an alternative source of 1,2-diacylglycerol, which activates isoforms of protein kinase C. The members of the protein kinase C family respond differently to various combinations of Ca²⁺, phosphatidylserine, molecular species of 1,2-diacylglycerol and other membrane phospholipid metabolites including free fatty acids. Protein kinase C isozymes are responsible for phosphorylation of specific cardiac substrate proteins that may be involved in regulation of cardiac contractility, hypertrophic growth, gene expression, ischemic preconditioning and electrophysiological changes. The initial product of phospholipase D, phosphatidic acid, may also have a second messenger role. As in other tissues, the question how the activity of phospholipase D is controlled by agonists in myocardium is controversial. Agonists, such as endothelin-1, atrial natriuretic factor and angiotensin 11 that are shown to activate phospholipase D, also potently stimulate phospholipase C- in myocardium. PMA stimulation of protein kinase C inactivates phospholipase C and strongly activates phospholipase D and this is probably a major mechanism by which agonists that promote phosphatidyl-4,5-bisphosphate hydrolysis secondary activate phosphatidylcholine-hydrolysis. On the other hand, one group has postulated that formation of phosphatidic acid secondary activates phosphatidyl-4,5-bisphosphate hydrolysis in cardiomyocytes. Whether GTP-binding proteins directly control phospholipase D is not clearly established in myocardium. Phospholipase D activation may also be mediated by an increase in cytosolic free Ca²⁺ or by tyrosine-phosphorylation.     

Biotransformation of monoterpenes and sesquiterpenes by cell suspension cultures of Achillea millefolium L. ssp. millefolium

The transformation capacity of Achillea millefolium L. ssp. millefolium (yarrow) cell suspension cultures was investigated using geraniol (50mg/l) and borneol, menthol, thymol and farnesols (25mg/l) as substrates. Apart from converting these substrates into several biotransformation products, the cell suspension cultures were also able to glycosylate both the substrates and the biotransformation products. aa]Key Words bb]Achillea millefolium L. ssp. millefolium bb]Yarrow bb]Compositae bb]Biotransformation bb]Glycosylation bb]Geraniol bb]Borneol bb]Menthol bb]Thymol bb]Farnesols     

Molecular analysis of a pistil-specific gene expressed in the stigma and stylar cortex of Solanum tuberosum

A gene, sts14, coding for a highly expressed mRNA in pistils of Solanum tuberosum, was isolated. Northern blot and in situ analyses demonstrated that the gene was expressed throughout pistil development in both the stylar cortex and the stigma. The deduced STS14 protein displays similarity to the pathogenesis-related PR-1 proteins. A possible function for protection or guidance of the pollen tubes through the pistil is discussed.     

Seasonal incidence of Ixodes ricinus ticks (Acari: ixodidae) on rodents in western France

Data collected from a longitudinal survey carried out over 2 years on four farms in western France were used to assess the incidence and infestation of Ixodes ricinus on rodents. Once a month, on each farm, 25 Sherman live traps were set in hedges bordering selected pastures. A total of 799 micromammals were examined, including Apodemus sylvaticus, Clethrionomys glareolus, Microtus agrestis, Microtus arvalis, and Crocidura spp. Larvae and nymphs of I. ricinus were found. Small numbers of Ixodes (Exopalpiger) trianguliceps were also recovered from each farm. The mean infestation rate of the I. ricinus larvae (1.6–5.9) among all animals examined varied between farms Most animals were infested by only a single tick, but one M. agrestis harboured 43 I. ricinus larvae. Larvae or nymphs were found throughout the year, with peaks from March to October.     

In vitro culture selection increases glyphosate tolerance in barley

In vitro culture of barley calluses has been used to produce plants with increased glyphosate tolerance. Calluses from immature embryos of barleyHordeum vulgare L. (Jeff) were cultured on Murashige and Skoog medium with 10^-6, 10^-5, 10^-4, 5×10^-4, 10^-3, or 10^-2M glyphosate for one, four or thirty months. Plants were regenerated from calluses maintained in glyphosate medium at 10^-6, 10^-5 or 10^-4M for four months, at 10^-5 or 5×10^-4M for one month and at 10^-5M for thirty months. The progeny of each regenerated plant was analyzed for response to glyphosate. Some progenies showed increased tolerance to glyphosate.     

Effects of gravitropic stress on the development of the primary root of lentil seedlings grown in space

Root growth and cell differentiation were analysed in lentil seedlings grown (1) in microgravity (F microg), (2) on the 1 x g centrifuge (F1 x g), (3) in microgravity and placed on the 1 x g centrifuge for 4 h [F(microg + 1 x g)], (4) on the 1 x g centrifuge and placed in microgravity for 4 h [F(1 x g + microg)]. In microgravity, there were strong oscillations of the root tip, even when the seedlings were grown first on the 1 x g centrifuge [F(1 x g + microg)]. In the [F(microg + 1 x g)] sample, the roots grown in microgravity were oblique with respect to the 1 x g acceleration when the seedlings were placed on the centrifuge. They were therefore gravistimulated. However, root length was similar in the 4 samples after 29 h of growth and growth rate of the root was the same between 25 h and 29 h although it appeared to be slightly greater in the [F(microg + 1 x g)] sample. Cell elongation was analysed as a function of the distance from the root cap junction. Cell length was similar in the seedlings grown in microgravity or on the 1 x g centrifuge. The transfer from the 1 x g centrifuge to microgravity [F(1 x g + microg)] did not modify cell elongation in the roots. Cell length in the roots which were grown in microgravity and gravistimulated [F(microg + 1 x g)] was different from that observed in microgravity but this was only due to gravistimulation. Thus, gravity does not have an effect on cell elongation when the roots are strictly oriented in the vertical position but it does as soon as the root tip deviates from this orientation.     

Dynamics of energy and nutrient concentration and construction cost in a native and two alien C4 grasses from two neotropical savannas

In Venezuela, the alien grasses Melinis minutiflora Beauv. and Hyparrhenia rufa (Nees.) Stapf tend to displace the native savanna plant community dominated by Trachypogon plumosus (Humb. and Bonpl.) Nees. This occurs in either relatively wetter and fertile highland savannas or in drier and less fertile lowland savannas. Although the native and aliens are perennial C₄ grasses, higher net assimilation leaf biomass per plant and germination rate of the latter are some causes for their higher growth rates and for their competitive success. The objective of this study is to compare seasonal tissue energy, N, P and K concentrations and the calculated construction costs (CC) between the native grass and either one of the alien grasses from lowland and highland savannas. We predict that, in order to out-compete native plants, alien grasses should be more efficient in resource use as evidenced by lower tissue energy and nutrient concentrations and CC.Tissue energy and nutrient concentration were measured throughout the year and compared between M. minutiflora and the co-occurring local population of T. plumosus in a highland savanna and between H. rufa and its neighbor local population of T. plumosus in a lowland savanna. CC was calculated from energy, N and ash concentrations considering ammonium as the sole N source. Differences between co-occurring species, T. plumosus populations, seasons, and organs were analyzed with ANOVA.Highland and lowland grasses differed in concentration and allocation of energy and nutrients whereas the differences between alien and native grasses were specific for each pair considered. Highland grasses had higher energy, N, P and CC than lowland grasses. These variables were always lowest in the culms. In the more stressed lowland site, tissue energy and nutrient concentrations decreased significantly during the dry season except in the roots of both grasses which had the highest energy and nutrients concentrations during the drought. This seasonal response was more marked in the local lowland population of T. plumosus in which maximum CC alternated seasonally between leaves and roots. Energy and nutrient concentrations and CC were the lowest in H. rufa. In the lowland savannas, the higher efficiency of resource use in the invader grass contributes to its higher competitive success through increased growth rate. In the highlands, overall tissue energy concentration and CC, but not N nor P concentration, were lower in the fast growing M. minutiflora but seasonal differences were lacking. The higher leaf CC in T. plumosus can be attributed to the higher proportion of sclerenchyma tissue which is more expensive to construct. Considering CC, both fast growing alien grasses are more efficient in resource use than the co-occurring native grass. However, the role of CC explaining the competitive success of the former, through higher growth rates, is more evident in the more stressful environment of the lowland savanna.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Germline genomic structure of the B10.A mouseTcra-V2 gene subfamily

 The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents. Received: 8 August 1995 / Revised: 24 April 1996