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.     

Limits to Catalysis by Ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O(5') bond in RNA. Although this enzyme has been the object of much landmark work in bioorganic chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k(cat)/K(m) for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k(cat)/K(m) for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC(6)H(4)-p-NO(2) by wild-type RNase A were found to be independent of glycerol concentration. Yet, UpA cleavage by the wild-type and mutant enzymes was found to have the same dependence on sucrose concentration, indicating that catalysis of UpA cleavage by RNase A is limited by desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k(cat) = 1.4 × 10(3) s(-1) and k(cat)/K(m) = 2.3 × 10(6) M(-1)s(-1) at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6-(3)H]Up[3,5,8-(3)H]A cleavage was found to be k(uncat) = 5 × 10(-9) s(-1) (t(1/2) = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10(11)-fold by binding to the transition state for P-O(5') bond cleavage with a dissociation constant of <2 × 10(-15) M.     

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.     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.     

Circadian and seasonal variation of the body temperature of sheep in a tropical environment

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55° C at 0500 hours in summer, and 38.87° C at 0600 hours in winter. The maximum was 40.03° C in summer (1700 hours) and 39.33° C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Spatio-temporal distribution of the microphytobenthic biomass in intertidal flats of Tagus Estuary (Portugal)

A study on spatio-temporal distribution of microphytobhethos in intertidal zones of Tagus Estuary was carried out from 1990 to 1992. Near Lisbon, Portugal, Tagus Estuary is a shallow mesotidal estuary, covering an area of 320 km². The intertidal area ranges from 20 to 40% off the total area and it is constituted mainly by mudflats. Intertidal flats are richly populated by microalgae, diatoms being the most important and ubiquitos group.Spatial variation of microphytobethos was studied in spring 1990, 21 different sites were sampled. Microphytobenthos biomass was evaluated as chlorophyll a content of the surface centimeter, ranging from 10 to 240 mg m^–2. A Principal Component Analysis showed that 62% of the total variability found in intertidal flats of Tagus estuary could be attributed to two major factors: sediment type and tidal height. A hierarchical grouping defined 3 major groups of similar stations, each one representing a different strata of the ecosystem.One station from each group was chosen for the study of the temporal variation. A sampling, rogram took place from April 1991 to April 1992, with fortnightly sampling, the Chl a ranged from 20–300 mg m^–2. No clear seasonal variation was found, and our results indicated that tidal height of sampledsite played an essential role in temporal biomass evolution, thus upper littoral sites were influenced by climatic parameters, whereas in lower sites action of tides mainly controlled microphytic biomass.

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Résumé Une étude sur l'hétérogénéité spatio-temporelle du microphytobenthos dans les sédiments intertidaux de l'Estuaire du Tage a été accompli de 1990 á 1992.L'Estuaire du Tage, prés de Lisbonne (Portugal) est un estuaire peu profond, mesotidal, avec une aire total de 320 km². L'aire intertidale est comprise entre 20 et 40% du total, et constituéé surtout par des vasiéres. Ces slikkes sont peuplées par une communauté assez riche de microalgues, ou les diatomées sont les plus abundantes.La variation spatialle du microphytobenthos était évalué au Printemps 1990, ou 21 différentes stations étaient échantillonnées. La biomasse était évalué par la concentration enchlorophylle a du premier centimétre de sédiment, qui a varié de 10 á 240 mg Chl a m^–2. Une Analyse en Composants Principales a montré que 62% de la variabilité de la biomasse était lié á deux facteurs: le sédiment et l'hauteur vis-á-vis la marée. Une classification hiérarchique des stations par similitude a établi 3 groupes principaux, représantantles différents strates de écecosytéme.Une station de chaque groupement a été choisie pour l'étude de la variation temporelle, qui s'est deroulé d'avril 1991 á avril 1992, avec des prélévements deux fois par mois. Les valeurs de Chl a obtenus vont de 20 á 300 mg m^–2. Les variations saisonniéres observées ne sont pas claires: nos résultats indiquent que l'hauteur de la station (m) joue un rôle essentiel dans l'évolution temporel de la biomasse, c'est á dire, la biomasse microalgal des sites du supra-littoral est influencié par les paramétres climatiques, tandis que dans l'infra-littoral c'est l'action des marées le facteur principal.

     

Linkage Analysis with an Alternative Formulation for the Mixed Model of Inheritance: The Finite Polygenic Mixed Model

This paper presents an extension of the finite polygenic mixed model of FERNANDO et al. (1994) to linkage analysis. The finite polygenic mixed model, extended for linkage analysis, leads to a likelihood that can be calculated using efficient algorithms developed for oligogenic models. For comparison, linkage analysis of 5 simulated 4021-member pedigrees was performed using the usual mixed model of inheritance, approximated by HASSTEDT (1982), and the finite polygenic mixed model extended for linkage analysis presented here. Maximum likelihood estimates of the finite polygenic mixed model could be inferred to be closer to the simulated values in these pedigrees.