Genetic transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> by particle bombardment

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe).     

Structural dissection of a highly knotted peptide reveals minimal motif with antimicrobial activity

The increasing occurrence of bacterial resistance to antibiotics is driving a renewed interest on antimicrobial peptides, in the hope that understanding the structural features responsible for their activity will provide leads into new anti-infective drug candidates. Most chemical studies in this field have focused on linear peptides of various eukaryotic origins, rather than on structures with complex folding patterns found also in nature. We have undertaken the structural dissection of a highly knotted, cysteine-rich plant thionin, with the aim of defining a minimal, synthetically accessible, structure that preserves the bioactive properties of the parent peptide. Using efficient strategies for directed disulfide bond formation, we have prepared a substantially simplified (45% size reduction) version with undiminished antimicrobial activity against a representative panel of pathogens. Analysis by circular dichroism shows that the downsized peptide preserves the central double alpha-helix of the parent form as an essential bioactive motif. Membrane permeability and surface plasmon resonance studies confirm that the mechanism of action remains unchanged.     

Functional-cranial approach to the influence of economic strategy on skull morphology

Environmental factors are assumed to play an important role in the shaping of craniofacial morphology. Here we propose a statistical approach which can be of utility in estimating the magnitude and localization of a particular nongenetic factor upon the specific functional components of the skull. Our analysis is a combination of previous attempts of apportionment of variance and the application of craniofunctional theory. The effect of subsistence strategy on craniofacial functional components was studied on 18 populations of hunter-gatherers and farmers from South America. Results demonstrate that the environmental factors studied likely influenced the masticatory component's size and shape. Even when this effect is not large enough to clearly differentiate among subsistence strategies (since whole craniofacial variation among populations remains greater), the method used here provides interesting clues to localize plastic or adaptive responses to external stimuli.     

Redox equilibrium in mucosal T cells tunes the intestinal TCR signaling threshold

Mucosal immune tolerance in the healthy intestine is typified by lamina propria T cell (LPT) functional hyporesponsiveness after TCR engagement when compared with peripheral blood T cell (PBT). When LPT from an inflamed intestine are activated through TCR cross-linking, their responsiveness is stronger. LPT are thus capable of switching from a tolerant to a reactive state, toggling between high and low thresholds of activation. We demonstrate that in normal LPT global tyrosine phosphorylation upon TCR cross-linking or an increase in intracellular H2O2, an inhibitor of protein tyrosine phosphatases, is muted. Thus, we propose that LPT have a greater reducing capacity than PBT, shifting the balance between kinases and protein tyrosine phosphatases in favor of the latter. Surface gamma-glutamyl transpeptidase, an indirect indicator of redox potential, and glutathione are significantly elevated in LPT compared with PBT, suggesting that elevated glutathione detoxifies TCR-induced reactive oxygen species. When glutathione is depleted, TCR-induced LPT tyrosine phosphorylation rises to PBT levels. Conversely, increasing glutathione in PBT attenuates tyrosine phosphorylation. In LPT isolated from inflamed mucosa, TCR cross-linking induces greater phosphorylation, and gamma-glutamyl transpeptidase levels are reduced compared with those from autologous noninflamed tissue. We conclude that the high TCR signaling threshold of mucosal T cells is tuned by intracellular redox equilibrium, whose dysregulation may mediate intestinal inflammation.     

Transferrin's mechanism of interaction with receptor 1

The kinetics and thermodynamics of the interactions of transferrin receptor 1 with holotransferrin and apotransferrin in neutral and mildly acidic media are investigated at 37 degrees C in the presence of CHAPS micelles. Receptor 1 interacts with CHAPS in a very fast kinetic step (<1 micros). This is followed in neutral media by the interaction with holotransferrin which occurs in two steps after receptor deprotonation, with a proton dissociation constant (K(1a)) of 10.0 +/- 1.5 nM. The first step is detected by the T-jump technique and is associated with a molecular interaction between the receptor and holotransferrin. It occurs with a first-order rate constant (k(-1)) of (1.6 +/- 0.2) x 10(4) s(-1), a second-order rate constant (k(1)) of (3.20 +/- 0.2) x 10(10) M(-1) s(-1), and a dissociation constant (K(1)) of 0.50 +/- 0.07 microM. This step is followed by a slow change in the conformation with a relaxation time (tau(2)) of 3400 +/- 400 s and an equilibrium constant (K(2)) of (4.6 +/- 1.0) x 10(-3) with an overall affinity of the receptor for holotransferrin [(K'1)(-1)] of (4.35 +/- 0.60) x 10(8) M(-1). Apotransferrin does not interact with receptor 1 in neutral media, between pH 4.9 and 6, it interacts with the receptor in two steps after a receptor deprotonation (K(2a) = 2.30 +/- 0.3 microM). The first step occurs in the range of 1000-3000 s. It is ascribed to a slow change in the conformation which rate-controls a fast interaction between apotransferrin and receptor 1 with an overall affinity constant [(K(3))(-1)] of (2.80 +/- 0.30) x 10(7) M(-1). These results imply that receptor 1 probably exists in at least two forms, the neutral species which interacts with holotransferrin and not with apotransferrin and the acidic species which interacts with apotransferrin. At first, the interaction of the neutral receptor with holotransferrin is extremely fast. It is followed by the slow change in conformation, which leads to an important stabilization of the thermodynamic structure. In the acidic media of the endosome, the interaction of apotransferrin with the acidic receptor is sufficiently strong and rate-controlled by a very slow change in conformation which allows recycling back to the plasma membrane.     

Spermiogenesis and ultrastructure of the spermatozoon of the liver fluke Fasciola gigantica Cobbold, 1856 (Digenea: Fasciolidae), a parasite of cattle in Senegal

The present paper describes the spermiogenesis and the ultrastructure of the spermatozoon of Fasciola gigantica, as revealed by transmission electron microscopy. Spermiogenesis in F. gigantica begins with the formation of a differentiation zone containing 2 centrioles with associated striated roots and an intercentriolar body between them. Each centriole develops a flagellum. Proximodistal fusion of these flagella with the median cytoplasmic extension gives rise to the spermatozoon. Spermiogenesis in F. gigantica is characterized by the formation of a dorsolateral cytoplasmic expansion, an external ornamentation of the cell membrane, and spinelike bodies. These 3 structures were also observed in the anterior part of the spermatozoon. Our study describes for the first time the simultaneous presence of dorsolateral cytoplasmic expansion, external ornamentation of the plasma membrane, and spinelike bodies in the spermatozoon of a trematode.     

Phenotypic evolution of human craniofacial morphology after admixture: a geometric morphometrics approach

An evolutionary, diachronic approach to the phenotypic craniofacial pattern arisen in a human population after high levels of admixture and gene flow was achieved by means of geometric morphometrics. Admixture has long been studied after molecular data. Nevertheless, few efforts have been made to explain the morphological outcome in human craniofacial samples. The Spanish-Amerindian contact can be considered a good scenario for such an analysis. Here we present a comparative analysis of craniofacial shape changes observed between two putative ancestor groups, Spanish and precontact Aztecs, and two diachronic admixed groups, corresponding to early and late colonial periods from the Mexico's Central Valley. Quantitative shape comparisons of Amerindian, Spanish, and admixed groups were used to test the expectations of quantitative genetics for admixture events. In its simplest form, this prediction states that an admixed group will present phenotypic values falling between those of both parental groups. Results show that, in general terms, although the human skull is a complex, integrated structure, the craniofacial morphology observed fits the theoretical expectations of quantitative genetics. Thus, it is predictive of population structure and history. In fact, results obtained after the craniofacial analysis are in accordance with previous molecular and historical interpretations, providing evidence that admixture is a main microevolutionary agent influencing modern Mexican gene pool. However, expectations are not straightforward when moderate shape changes are considered. Deviations detected at localized structures, such as the upper and lower face, highlight the evolution of a craniofacial pattern exclusively inherent to the admixed groups, indicating that quantitative characters might respond to admixture in a complicated, nondirectional way.     

Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi

Activation of virulence in pathogenic fungi often involves differentiation processes that need the reset of the cell cycle and induction of a new morphogenetic program. Therefore, the fungal capability to modify its cell cycle constitutes an important determinant in carrying out a successful infection. The dimorphic fungus Ustilago maydis is the causative agent of corn smut disease and has lately become a highly attractive model in addressing fundamental questions about development in pathogenic fungi. The different morphological and genetic changes of U. maydis cells during the pathogenic process advocate an accurate control of the cell cycle in these transitions. This is why this model pathogen deserves attention as a powerful tool in analyzing the relationships between cell cycle, morphogenesis, and pathogenicity. The aim of this review is to summarize recent advances in the unveiling of cell cycle regulation in U. maydis. We also discuss the connection between cell cycle and virulence and how cell cycle control is an important downstream target in the fungus-plant interaction.