Intracellular signaling following myocardial stretch: an autocrine/paracrine loop

The stretch of adult papillary muscle elicits a chain of autocrine/paracrine events in which the Na(+)/H(+) exchanger (NHE-1) activation is the central step. This activation is induced by a sequential angiotensin II-endothelin (Ang II-ET) release and results in an increase in intracellular Na(+) (Na(+)(i)) without significant changes in intracellular pH. The increase in Na(+)(i) negatively shifts the reverse potential of the Na(+)/Ca(2+) exchanger (NCX) thus inducing cell Ca(2+) influx that augments myocardial contractility. This increase in force represents the mechanical counterpart of the autocrine/paracrine mechanism triggered by stretch and has been called the slow force response (SFR) to stretch.     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

Geography influences microsatellite polymorphism diversity in Amerindians

Data related to 15 short tandem repeat polymorphisms (STRPs) are reported for four South American Indian populations, and integrated with previous Brazilian Indian results. Overall heterozygosities varied significantly among groups (Kruskal-Wallis test, P = 0.002). The lowest levels of heterozygosity were observed in the Ache, Ayoreo, and Surui, an expected finding considering their isolation and ethnohistory. Genetic distance and gene diversity analyses suggested that geography was a good predictor of genetic affinity among these Native Americans. New evidence from this study supports the hypothesis that the Ache population descends from a Ge group that preceded the Guarani colonization of Paraguay.     

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.     

Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study

Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.     

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.     

The public health implications of maternal care trade-offs

The socioeconomic and ethnic characteristics of parents are some of the most important correlates of adverse health outcomes in childhood. However, the relationships between ethnic, economic, and behavioral factors and the health outcomes responsible for this pervasive finding have not been specified in child health epidemiology. The general objective of this paper is to propose a theoretical approach to the study of maternal behaviors and child health in diverse ethnic and socioeconomic environments. The specific aims are: (a) to describe a causal pathway between the utility that women obtain through work outside the home and through child care and disease hazard rates in childhood using an optimization model; (b) to specify the influence of ethnic and socioeconomic factors on model constraints; (c) to use the model as a tool to learn about how different combinations of maternal wage labor and child care time might influence child health outcomes in diverse social contexts; (d) to identify parameters that will require measurement in future research; (e) to discuss research strategies that will enable us to obtain these measurements; and (f) to discuss the implications of the model for biostatistical modeling and public health intervention. Optimization models are powerful heuristic tools for understanding how ethnic, environmental, family, and personal characteristics can place important constraints on both the quality and quantity of care that women can provide to their children. They provide a quantitative appreciation for the difficult trade-offs that most women face between working in order to purchase basic goods that children cannot do without (e.g., food, clothing, shelter, health insurance), and increasing offspring well-being through child care (e.g., training in social skills, affection, protection from environmental hazards, help with homework). The research was funded by a Faculty Scholars Award from the William T. Grant Foundation to A. Magdalena Hurtado. A. Magdalena Hurtado, Ph.D., is an associate professor in the Department of Anthropology, University of New Mexico. Her research interests include the origins of the sexual division of labor, epidemiology of indigenous peoples, disease susceptibility, the development and intergenerational transmission of antigens and immune defense, immune function and allergic sensitization, and trauma. She also works on public health interventions, biological capital and poverty, and land tenure and human rights in native communities of South America. Carol Lambourne, M.Sc., is a doctoral candidate in the Department of Anthropology, University of New Mexico. Her research interests are evolutionary models of child and adolescent development, life history theory, family composition and investment patterns, pubertal timing and psychosexual maturation, juvenile stress, and infanticide. Kim Hill, Ph.D., is a professor in the Department of Anthropology, University of New Mexico. His research interests are modern hunter-gatherers, including extensive fieldwork in lowland South America. Current topics of interest include human evolution, economic strategies, life history theory, the evolution of cooperation, and the emergence of social norms enforced by punishment. He is also involved in economic development, health and education projects with lowland South American native populations. Karen Kessler received her M.S. in Anthropology from the University of New Mexico in 1996. Her research interests are the application of mathematical modeling to the prevention of diabetes and other causes of morbidity and mortality in historical populations.     

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.     

Role of protein phosphatases 2C on tolerance to lithium toxicity in the yeast Saccharomyces cerevisiae

Protein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild-type cells and that this is most probably the result of increased expression of the ENA1 Na(+)-ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium-sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1-4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li(+) and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.     

Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.