An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of ¹²⁵ I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10^?9 M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10⁶ per cell of 5–7 fissions of age, to about 16 × 10⁶ at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from “immature” to “adult,” that is competent to respond as well to pheromones of conspecific, genetically different cells. © 1992 Wiley-Liss, Inc.     

Enzyme polymorphism in a population of Calomys musculinus (Rodentia,Cricetidae)

NAD-linked lactate, malate, glycerophosphate, alcohol and nonspecific dehydrogenases, aspartate aminotransferases, and soluble esterases from extracts of tissues of individuals from a wild population of Calomys musculinus (Rodentia, Cricetidae) have been analyzed by means of starch gel electrophoresis and specific staining. Allelic frequencies and heterozygosity have been determined. Mendelian inheritance of some of the variants detected was confirmed by breeding experiments. Ten out of fifteen (66.6%) of the genetic loci investigated presented polymorphism. Mean heterozygosity per locus was very high (H=0.2014, se 0.046).This work has been supported, in part, by grants from the Secretaria de Ciencia y Tecnología de la Nación (National Program for Endemic Diseases) and from the Fundación Emilio Ocampo. C. N. G. is a Fellow and A. B. a Career Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.     

Review of the Neotropical Isertieae (Rubiaceae)

All neotropical genera previously included in the tribe Isertieae A. Rich. ex DC. are considered. The tribe Isertieae is here delimited by the following characters: absence of rhaphides, valvate aestivation of the corolla, cordiform or discoid placenta, fleshy indehiscent fruit, and seed coat cells with pitted walls. As so delimited, the tribe definitely includes the neotropical generaIsertia Schreb.,Gonzalagunia Ruiz & Pav.,Amphidasya Standl.,Raritebe Wernh.,Sabicea Aubl., andMussaenda L. as well as some paleotropical genera not here considered. The following neotropical genera are excluded from tribe Isertieae:Hippotis Ruiz & Pav.,Pentagonia Benth.,Sommera Schlecht.,Tammsia Karst.,Coccocypselum P. Browne,Schradera Vahl,Didymochlamys Hook, f., andXerococcus Oerst. The position of four genera,Striolaria Ducke,Patima Aubl.,Habroneuron Standl., andNeosabicea Wernh., remains uncertain due to a lack of material.     

Chromosome polymorphism in populations of the grasshopper Trimerotropis pallidipennis from southern Argentina

Three populations of the grasshopper Trimerotropis pallidipennis from southern Argentina have been studied cytologically. A very characteristic B-chromosome was found in all three. They also showed geographical variability in respect of the presence of pericentric inversions, and the inversion system was found to influence chiasma frequency. The Laguna Blanca population, which is on the hypothetical pathway the species is believed to have followed during its migration from northern to southern latitudes, has the same karyotype composition as the N. American form, with fixed inversions in the 3 largest autosomes and the X-chromosome. Its members have a high total chiasma frequency and a great number of interstitial chiasmata. The Sierra de la Ventana population, situated at the absolute eastern border of the species distribution is highly polymorphic with respect to the presence of inversions in the medium chromosomes. Its members have the lowest total chiasma frequency and a greatly reduced number of interstitial chiasmata. Situated geographically between the other two, the Choele-Choel population has the highest frequency of inversions and many of them are homozygous. Its members have a higher total chiasma frequency than that observed in specimens from Sierra de la Ventana, and a greatly reduced number of interstitial chiasmata, similar to that observed in individuals from the latter population.     

Human urine: Epicatechin metabolites and antioxidant activity after cocoa beverage intake

Associations between cocoa consumption in humans, excreted metabolites and total antioxidant capacity (TAC) have been scarcely investigated. The aims of the study were to investigate the epicatechin (( ? )-Ec) metabolites excreted in urine samples after an intake of 40 g of cocoa powder along with the TAC of these urine samples and the relation between both the analyses. Each of the 21 volunteers received two interventions, one with a polyphenol-rich food (PRF) and one with a polyphenol-free food (PFF) in a randomized cross-over study. Urine samples were taken before and during 24 h at 0–6, 6–12 and 12–24 h periods after test intake. The excreted ( ? )-Ec metabolites and the TAC were determined in urine samples by LC-MS/MS and TEAC assay, respectively. The maximum excretion of ( ? )-Ec metabolites and the maximum TAC value were observed in urine samples excreted between 6 and 12 h after PRF consumption. Significance of TAC increase was found in urine samples excreted during 0–6 and 6–12 h (66.6 and 72.67%, respectively, with respect to the 0 h).     

An Examination of Morphometric Variations in a Neotropical Toad Population (Proceratophrys cristiceps,Amphibia, Anura,Cycloramphidae)

The species Proceratophrys cristiceps belongs to the genus Proceratophrys within the family Cycloramphidae. These amphibians are found exclusively in South America in the morphoclimatic domain of the semi-arid depression zones in northeastern Brazil known as the Caatinga. We examined intrapopulational variation using univariate and multivariate statistics with traditional and geometric morphometrics, which supported the existence of two morphotypes of this species. Our results indicated significant degrees of variation in skeletal characteristics between some natural populations of this species. Careful analyses of variability levels are fundamental to avoid taxonomic errors, principally in populations that demonstrate characteristics intimately associated with their area of occurrence, as is the case of Proceratophrys cristiceps.