Synthesis and β-conformation of sequential polypeptides containing arginine,histidine, and leucine

Alternating poly(Arg-Leu) and copolypeptides with Arg-Leu and His-Leu sequences were prepared by condensation of the corresponding p-nitrophenyl dipeptide esters in the presence of 1-hydroxybenzotriazole. Arginine was used without any protection and histidine side chains were protected using π-benzyloxymethyl group recently introduced in peptide chemistry. The ability of these polypeptides with alternating hydrophilic and hydrophobic residues to form water-soluble β-sheets was investigated by CD.     

Chemical activity of simple basic peptides

Alternating all-L poly(leucyl-lysyl) increases markedly the rate of hydrolysis of oligoribonucleotides. Pure D poly (leucyl-lysyl) is as active as the all-L polymer. The homochiral polypeptides adopt a-sheet structure when complexed to the oligonucleotides. Alternating poly(D,L-Leu-D,L-Lys) made of racemic amino acids is much less efficient and is unable to adopt a-sheet structure. A set of alternating poly (leucyl-lysyl) ranging from the racemic to the homochiral all-L polymer has been checked. Their conformations can be described as a mixture of random coil and-sheet conformations, the amount of-sheet increasing with the optical purity of the polymer. The hydrolytic activity follows the proportion of-sheets, suggesting that the chemical activity is related to the geometry of the chain. Short peptides were prepared in order to evaluate the critical chain length required for the hydrolytic activity. A decapeptide is long enough to present 90% of the activity of the corresponding polypeptide.     

Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus

Summary Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants.Abbreviations 2,4D 2,4 dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - CP coat protein - EDTA ethylene diamine tetraacetic acid - ELISA enzyme-linked immunosorbent assay - GFLV grapevine fanleaf virus - GUS glucuronidase - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - NOA -naphthoxyacetic acid - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Logné, a peat bog of European ecological interest in the Massif Armorican, Western France: Bog development, vegetation and land-use history

Logné, one of three raised bogs (Hochmoor) in the Massif Armorican, was officially designated as a nature reserve in 1987 and is of considerable ecological interest from botanical, ornithological and herpetological aspects. Palynological investigations reveal the history of local and regional vegetation during the last ca. 4000 years. From the end of the Neolithic Age to the present day, changes in the bog appear to have been considerably influenced by variations in local watertable level that may be of natural origin or caused by human activity. Alder carr withOsmunda regalis, which was the dominant vegetation around the periphery of the bog at ca. 4200 B.P., underwent many changes. Its final demise occurred in Gallo-Roman times when the local landscape took on a distinctly open appearance. The first clear evidence for cereal cultivation relates to the Bronze Age. After a decline in farming at the beginning of the Iron Age, there was a strong renewal of farming in the La Tène period, which included a distinct arable component and also records forJuglans andCastanea pollen. The timing of the introduction of walnut and chestnut to the Massif Armorican is discussed.     

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.     

Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry

This study aimed at establishing a sensitive multiple reaction monitoring‐mass spectrometry (MRM‐MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM‐MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM‐MS measurement were also determined. The method was applied to liver samples from ten non‐cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM‐based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM‐MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

The sterols of Dynamena pumila

The sterols of the hydroid, Dynamena pumila (Sertulariidae) have been analysed for the first time. Results from TLC, GLC, mass spectrometry and NMR have led to the determination of the structure of 16 steroids, the main being cholesterol (37%). A series of Δ_{5 22} C₂₆, C₂₇, C₂₈ C₂₉ sterols, and the corresponding stanols and fucostanol, have also been identified.