Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Rainfall and flowering synchrony in a tropical shrub: Variable selection on the flowering time ofErythroxylum havanense

Summary We tested the adaptive significance of flowering synchrony by means of a quantitative analysis of selection and by flowering induction experiments with the deciduous shrubErythroxylum havanense. Temporal schedules of flower and fruit production were determined for a local population (in three sites) in a Mexican seasonal forest for 2 years (1987–1988). The consequences of natural variation in flowering time (flowering initiation day) on maternal reproductive success (fecundity) were evaluated. We observed high levels of inter- and intraindividual flowering synchrony in 1987, but not in 1988 and this contrast was related to differences in rainfall patterns between the two years. A significant proportion (15.4%) of the phenotypic variation in flowering initiation day was accounted for by environmental variance. The expression of phenotypic variance of flowering time and, consequently, the opportunity for selection to act, are controlled by annual variation in rainfall. Despite the between-year difference in flowering synchrony, we detected a relatively intense directional selection on flowering initiation day in both years, but selection coefficients were of opposite sign (standardized directional gradients were –0.326 and 0.333 for 1987 and 1988, respectively). For both years there was a significant relationship between individual relative fitness and the number of neighbouring flowering plants in a given day, suggesting positive frequency-dependent selection.     

Comparison of the effects of hydrophobicity,amphiphilicity, and α-helicity on the activities of antimicrobial peptides

Multiple linear regression was used to quantify the dependence of the antimicrobial activity of 13 peptides upon three calculated or experimentally determined parameters: mean hydrophobicity, mean hydrophobic moment, and α-helix content. Mean hydrophobic moment is a measure of the amphiphilicity of peptides in an α-helical conformation. Antimicrobial activity was quantified as the reciprocal of the measured minimal inhibitory concentration (MIC) against Escherichia coli. One of the peptides was magainin 2, and the remainder were novel peptides designed for this study. The multiple linear regression results revealed that the amphiphilicity of the peptides was the most important factor governing anti-microbial activity compared to mean hydrophobicity orα-helix content. A better regression cf the data was obtained using In(1/MIC + constant) as the dependent variable than with either 1/MIC or In(1/MIC). These results should be useful in designing peptides with higher antimicrobial activity. © 1995 Wiley-Liss, Inc.     

Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line.

The biosynthesis, post-translational modifications, and oligosaccharide structure of human CD8 glycoprotein have been studied in transfected rat epithelial cells. These cells synthesized and expressed on the plasma membrane high amounts of CD8 in a homodimeric form stabilized by a disulfide bridge. Three different CD8 forms were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after metabolic labeling and immunoprecipitation: a newly synthesized, unglycosylated 27-kDa (CD8u), a palmitylated and initially O-glycosylated 29-kDa (CD8i), and the mature, terminally glycosylated 32-34-kDa doublet (CD8m). CD8i is a transient intermediate form between CD8u and CD8m: characterization of carbohydrate moiety of [3H]glucosamine-labeled CD8i showed that it comprises for the vast majority non-elongated O-linked GalNAc closely spaced on the peptide backbone. Structural analysis of oligosaccharides released by mild alkaline borohydride treatment from the [3H]glucosamine-labeled CD8 34-kDa form showed that the neutral tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAcOH, and an homologous monosialylated pentasaccharide, predominate; the disialylated NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6) GalNAcOH tetrasaccharide appeared to be poorly present. In the CD8 32-kDa form the neutral tetrasaccharide was by far the prominent O-linked chain, and no disialyloligosaccharides were identified. These results indicate that the maturation of CD8 glycoprotein in transfected rat epithelial cells results in the formation of branched O-linked oligosaccharides and that a higher degree of sialylation is responsible for the production of the heavier 34-kDa form.     

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein

Journal of Physiology and Biochemistry - The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical...     

A partnership project for a new Red List of the Italian Flora

Abstract

The IUCN Red Lists assessment provides an internationally accepted system to verify the extinction risk of species. Working Groups of the Italian Botanical Society have recently discussed the importance of producing a reliable list of species at the national level. This list could be the starting point for future in situ and ex situ plant conservation activities.     

Iron Release in Erythrocytes and Plasma Non Protein-bound Iron in Hypoxic and Non Hypoxic Newborns

Iron is released in a desferrioxamine (DFO)-chelatable form (DCI) when erythrocytes are challenged by an oxidative stress. In &#103 -thalassemic erythrocytes, both DCI content and release (after aerobic incubation for 24 h) are increased and correlated with the fetal hemoglobin (HbF) levels. Since erythrocytes from newborns have an extremely high content of HbF and are exposed to conditions of oxidative stress, the release of iron in these erythrocytes was investigated. The erythrocyte DCI content was increased in preterm but not in term newborns as compared to adults, while the release was increased in both preterm and term erythrocytes. The level of plasma non protein-bound iron (NPBI), which was not detectable in adults, was much higher in preterm than in term newborns. When term plus preterm newborns were divided in two groups, normoxic and hypoxic, according to cord blood pH, it was found that both iron release and NBPI were markedly higher in the hypoxic newborns compared to normoxic ones. Similar results were also obtained when the preterm and term infants were considered separately on the basis of cord blood pH. Therefore, iron release and NPBI are higher when conditions of hypoxia occur. In fact, when the values for iron release and NPBI were separately plotted against cord blood pH values, significant negative correlations were seen in both cases. NPBI was correlated with iron release seen in all the newborns and a significant part of the released iron could be recovered into the incubation medium at the end of the incubation.