Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity.

Cathepsin D (CatD) is a member of the mammalian aspartic protease family and is involved in cellular protein degradation and in several pathological processes. A sensitive and specific assay for the determination of CatD activity in biological samples was developed. The peptide amide substrates Amca-EDKPILF downward arrowFRLGK(biotin)-CONH2 (I), Amca-EEKPIC(Acm)F downward arrowFRLGK(biotin)-CONH2 (II) and Amca-EEKPISF downward arrowFRLGK(biotin)-CONH2 (III) contain a CatD cleavage site (F downward arrowF) flanked by a N-terminal Amca-fluorophore (7-amino-4-methylcoumarin-3-acetic acid) and a C-terminal biotin moiety. Substrates II and III proved to be specific substrates containing only one cleavage site for CatD. After cleavage of the Phe-Phe bond by CatD all biotin conjugated peptides were removed with streptavidin-coated magnetic beads. The remaining fluorescent peptides in solution represent the amount of digested substrate. The versatility of this CatD digest and pull down assay was demonstrated by measuring the activity of CatD in different subcellular fractions of human EBV-transformed B cells and human monocytes. The described method based on the designed CatD substrates represents a valuable tool for routine assays.     

Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Host Location of a Pupal Parasitoid in a Tritrophic System Compared to a Model Offering Mechanosensory Cues Only

Several species of hymenopteran parasitoids are able to locate concealed pupal hosts by vibrational sounding. However, the specific role of this technique of mechanosensory host location has not yet been investigated in a natural, tritrophic system with multiple cues. Here we compared the host location of the pupal parasitoid Xanthopimpla stemmator in a tritrophic system with corn borer pupae in maize stem to the behavior on a paper model offering mechanosensory cues only. In general, the behavioral pattern and the behavioral states exhibited by host-searching female parasitoid were identical in the model and in the tritrophic system, while quantitative aspects differed. Our results demonstrate that vibrational sounding maintains its significance for host location in an environment with multiple cues, and that additional cues may increase responsiveness of females.     

Synthesis and reactivity of early-late heterobimetallic complexes displaying a η-tantalum-alkylidene to palladium interaction

Tantalaalkylidene compounds, CHR=TaCl₃L₂ (R=^tBu or CMe₂Ph, L=THF or 1/2dimethoxyethane) mixed with the cyclopalladated dimer [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)]₂, 1, afford good yields of heterodimetallic complexes [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CHR=TaCl₃L], 3a, 3b, in which the Ta=C unit is η²-interacting with the palladium atom, while a chloride ligand is bridging the tantalum and the palladium atoms. These compounds are fairly stable in air in the solid state and also in solution at RT. The interaction of the Ta=C unit with Pd in these bimetallic compounds is weak as shown by the ready formation of [Pd(2-C₆H₄CH₂NMe₂)PyCl] and CHR=TaCl₃Py₂ upon treatement with pyridine. Compounds analogous to 3a, b can also be obtained with 12 electrons tantalum complexes. Thus treating the same cyclopalladated dimer 1 with CHR=Ta(OAr)₃ (OAr=2,6-diisopropylphenyloxy) led to a much more stable though electron deficient species: [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=Ta(OAr)₃], 3c. Substitution in 3a of one chloride ion by an alkyl group occurred at the tantalum metal via reaction with ZnR₂ (R=---CH₂CMe₂Ph) leading to [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=TaCl₃(CH₂CMe₂Ph)], 4 for which there is no free rotation around the new Ta---C bond and in which one of the methylene protons is strongly interacting with the palladium centre. This compound is believed to mimic an intermediate to the formation of tantalacarbyne derivative, which was obtained earlier via reaction of the uncomplexed tantalacarbene compound with dialkylzinc compounds.     

A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum

The investigation of plant cytochrome P450 genes and enzymes is a field of growing interest. Apparently, an even greater diversity of cytochrome P450 genes exists in plants in comparison to other eukaryotes. This may be due to their role in the biosynthesis of secondary metabolites that are present in plants in an enormous variety. Most cloning approaches are hampered by the large sequence diversity of plant cytochrome P450 genes. We present a method to clone divergent cytochrome P450 ESTs by a nested RT-PCR-strategy. These ESTs were used for the subsequent cloning of the corresponding full-size cDNAs of divergent families via cDNA-library screening. Sixteen cytochrome P450 genes belonging to different cytochrome P450-families have been identified in this way, proving the efficacy of the strategy. Received: 1 December 2000 / Accepted: 26 February 2001     

Functional genomics of phosphate antiport systems of plastids

Plant cells require a co-ordination of metabolism between their major compartments, the plastids and the cytosol, in particular as certain metabolic pathways are confined to either compartments. The inner envelope membrane of the plastids forms the major barrier for metabolite exchange and is the site for numerous transport proteins, which selectively catalyse metabolite exchanges characteristic for green and/or non-green tissues. This report is focused on the molecular biology, evolution and physiological function of the family of phosphate translocators (PT) from plastids. Until now, four distinct subfamilies have been identified and characterized, which all share inorganic phosphate as common substrate, but have different spectra of counter exchange substrates to fulfil the metabolic needs of individual cells and tissues. The PTs are named after their main transported substrate, triose phosphate (TPT), phosphoenolpyruvate (PPT), glucose 6-phosphate (GPT) and xylulose 5-P (XPT). All PTs belong to the TPT/nucleotide sugar transporter (NST) superfamily, which includes yet uncharacterized PT homologues from plants and other eukaryotes. Transgenic plants or mutants with altered transport activity of some of the PTs have been generated or isolated. The analysis of these plant lines revealed new insights in the co-ordination and flexibility of plant metabolism.     

Invertébrés remarquables du Callovien inférieur de la Voulte-sur-Rhône (Ardèche, France)Remarkable Invertebrates from the Lower Callovian of la Voulte-sur-Rhône (Ardèche, France)

Remarkable Invertebrates from the Lower Callovian of la Voulte-sur-Rhône (Ardèche, France). The four-excavating field expeditions from 1983 to 1986 led to collect a considerable amount of new data from this exceptional paleontological site, which was neglected, and unexplored for the essential, until now. This paper presents a synthesis of the main available results. For the first time, the succession of the lithofacies is precised from all preserved deposits thick about 12 m. These observations reveal, on the whole, a great homogeneity of lithologic, paleobiologic or taphonomic characteristics, which denote a rather great constancy of the global environmental conditions. The lithofacies are schistous marls revealing former uncemented paleosubstrates, with clay very dominating (montmorillonite for 60–70%, calcium carbonate, siderite and limonite for less than 30% or 40%). The faunas are nearly exclusively composed of pelagic, nectopelagic or mesopelagic organisms (swimming in full water or dependent on free algal environments); they include very few benthic organisms. The marine water depth in this area would be estimated to have reached roughly 200 m. The surface and middle water strata, where lived the major part of organisms, must have been usually agitated, well oxygenated and illuminated. At the opposite side, the bottom water must have been very quiet and anoxic, in aphotic zona, which is responsible for the soft tissue mineralisation.     

Vitellogenin from female and estradiol-stimulated male river lampreys (Lampetra fluviatilis L.).

The influence of estradiol-17beta (E(2)) on vitellogenesis is well documented for a number of oviparous craniates. We have examined the role that estradiol-17beta plays in the induction and regulation of vitellogenin synthesis in the maturing European river lamprey, Lampetra fluviatilis. In both females and males the estradiol-17beta concentrations in the plasma reached comparable maximum values in March, only a few weeks before spawning. Throughout the spawning run, the vitellogenin titer in the blood of females remains rather constant while the ovary volume increases. In contrast, we never found circulating VTG in untreated male lampreys. The synthesis and secretion of the yolk precursor molecule can be induced in males, however, by high doses of estradiol injected into the coelom. Lamprey vitellogenin was isolated from the blood of maturing females as well as from hormone-stimulated males and identified by its immunological and electrophoretic properties. In the blood plasma of both maturing female and estradiol-treated male lampreys it always appears simultaneously in two different molecular forms: a vitellogenin monomer with an apparent molecular weight of 310-330kDa and a dimer. After SDS treatment, vitellogenin is represented as a 212-kDa polypeptide.     

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml^-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml^-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.