Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

Cyclodextrins enhance the aerobic degradation and dechlorination of low-chlorinated biphenyls

Summary The aerobic dechlorination of 4-chlorobiphenyl and of Aroclor 1221 by Pseudomonas sp. CPE1 strain was enhanced by the presence of hydroxypropyl--cyclodextrin in the medium. The best dechlorination results were obtained when 4-chlorobiphenyl or Aroclor 1221 and the cyclodextrin were used at the molar ratios 1:1 and 1:1.5. This agent can be adopted to enhance the efficiency of PCB degradation tests.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Synthesis and biological activity of tripeptide FR113680 analogues containing unconventional amino acids

In order to further develop structure–activity relationships and to get information about the biological active conformations we synthetized analogues tripeptide to the FR 113680 [Ac- Thr-D -Trp(CHO)-PheNMeBzl; Ac: acethyl], in which the phenylalanine residue was replaced by unconventional amino acids [1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); (3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic); (S,S,S)-2-azabiciclo[3.3.0]octane-3-carboxylic acid (Aoc); 3-(1′-naphthyl) alanine (Nap) phenylglicine (Phg); thienylalanine (Thi)]. The biological activity of the peptides was performed on guinea pig ileumfar neurokinin 1 (NK-1) and on rat colon for neurokinin 2 (NK-2). In particular, the replacement of the Phe³ by the Oic ( 8 _a) gave an higher antagonist activity in both NK-1 and NK-2 receptors, but no improvement in selectivity with respect to reference tripeptide (FR113680) The compound ( 8 _a) represent the first example of highly potent peptides that do not contain an aromatic mi no acid of the third position as had been previously considered essential. © 1995 John Wiley & Sons, Inc.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.