Receptor fragment approach to the binding between CCK8 peptide and cholecystokinin receptors: a fluorescence study on type B receptor fragment CCK(B)-R (352-379)

Fluorescence titrations in a membrane mimetic solvent system allowed us to estimate that the dissociation constant of the bimolecular complex between CCK8 peptide and cholecystokinin type B receptor fragment CCK(B)-R (352-379) is in the micromolar range. When considered in the context of the full receptor/ligand model, these experiments demonstrate that the receptor fragment chosen on the basis of previous structural studies represents a reliable model system to monitor the ability of CCK8 or CCK8 analogs to bind the cholecystokinin receptor. Together with previous studies, this confirms that the receptor fragment approach adopted to define the binding mode of the CCK8 fragment of cholecystokinin with its two receptors, CCK(A) and CCK(B,) can be used to characterize the binding from the equilibrium standpoint. In this context, fluorescence spectroscopy proves to be the favored technique to measure dissociation constants in the nanomolar to micromolar range.     

Metachromatic effects and binding of organic cations to energized submitochondrial particles

Energization of submitochondrial particles results in a marked increase of binding, measured as number of sites and binding constants, of the cationic dyes Acridine Orange and Neutral Red. The binding of the dyes is accompanied by spectral changes which are identified as metachromatic effects. The findings are interpreted in terms of interaction with electron-negative sites and stacking of the dye molecules.     

Phylogeny and infrageneric classification of Symplocos (Symplocaceae) inferred from DNA sequence data

Symplocos comprises ～300 species of woody flowering plants with a disjunct distribution between the warm-temperate to tropical regions of eastern Asia and the Americas. Phylogenetic analyses of 111 species of Symplocos based on the nuclear ribosomal internal transcribed spacer (ITS) region and the chloroplast genes rpl16, matK, and trnL-trnF yielded topologies in which only one of the four traditionally recognized subgenera (Epigenia; Neotropics) is monophyletic. Section Cordyloblaste (subgenus Symplocos; eastern Asia) is monophyletic and sister to a group comprising all other samples of Symplocos. Section Palura (subgenus Hopea; eastern Asia) is sister to a group comprising all other samples of Symplocos except those of section Cordyloblaste. Symplocos wikstroemiifolia (eastern Asia) and S. tinctoria (southeastern United States), both of subgenus Hopea, form a clade that groups with S. longipes (tropical North America) and the species of subgenus Epigenia. The remaining samples of subgenus Hopea (eastern Asia) form a clade. Section Neosymplocos (subgenus Microsymplocos; Neotropics) is well nested within a clade otherwise comprising the samples of section Symplocastrum (subgenus Symplocos; Neotropics). Section Urbaniocharis (subgenus Microsymplocos; Antilles) groups as sister to the clade comprising Symplocastrum and Neosymplocos. The data support the independent evolution of deciduousness among section Palura and S. tinctoria. The early initial divergence of sections Cordyloblaste and Palura from the main group warrants their recognition at taxonomic levels higher than those at which they are currently placed. An inferred eastern Asian origin for Symplocos with subsequent dispersal to the Americas is consistent with patterns from other phylogenetic studies of eastern Asian-American disjunct plant groups but contrary to a North American origin inferred from the earliest fossil occurrences of the genus.     

Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture

Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.     

Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterranean Sea)

A total number of 40 potentially toxic microalgae and 5 taxa causing discolorations have been identified along the coasts of the Campania region (South Tyrrhenian Sea, Mediterranean Sea). This number is based on results of over 20 years of research at a coastal station in the Gulf of Naples, 4 years of monitoring activity along the Campania coasts and of a series of scattered and sporadic observations. Several species of the recently erected genera Karenia and Takayama are reported for the first time in the area. Information on the period of highest abundances or most probable period of occurrence indicates the late spring and summer as the periods of maximum risk of harmful events. Despite the variety of potentially toxic species, no human health problems nor fish kills have ever been recorded in Campania. The reasons for this apparent paradox are probably to be found in the ecological factors that regulate the abundance, toxicity and spatial distribution of the potentially harmful species and reflect as well the relatively low number of aquaculture plants in the area.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

The Role of the Formamide/Zirconia System in the Synthesis of Nucleobases and Biogenic Carboxylic Acid Derivatives

We describe the one-pot synthesis of a large variety of nucleic acid bases and related compounds from formamide in the presence of zirconium minerals as catalysts. The major products observed are: purine, 2-hydroxy pyrimidine, 5-hydroxy pyrimidine, isocytosine, adenine, urea, and carbodiimide. The synthesis of low molecular weight amides and carboxylic acid derivatives (intermediates of extant metabolism) was also observed: glyoxylamide, glycolic-, lactic-, succinic-, oxalic-, fumaric-, and maleic acids. As the major problem in the origin of informational polymers is the instability of their precursors, we also investigated the effects of zirconia minerals on the stability of ribooligonucleotides in formamide and in water. The relevance of these findings with respect to the origin of informational polymers and primordial metabolism is discussed.