An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.     

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

Summary The adamantane moiety was introduced in the tachykinin NK₂ receptor-selective agonist [-Ala⁸]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂, MEN 10210) and in different positions of the NK₂ receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂) in order to investigate how this substitution affects their biological activity at tachykinin NK₁, NK₂ and NK₃ receptors. 1-Adamantaneacetic acid (1-Ada-CH₂COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH₂CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH₂CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH₂CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH₂CO)-NH₂]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK₁ and NK₂ receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK₃ receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK₁, NK₂ and NK₃ receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK₂ receptors of the rabbit isolated pulmonay artery.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

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.     

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.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Matric potential evaluations and measurements for gelled substrates

A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement.     

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.     

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.     

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m₃G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hibernating dormice, were present in much higher amouts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.