Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.     

Characterization of polymorphic microsatellite loci in Haageocereus (Trichocereeae, Cactaceae)

? Premise of the study: Microsatellites were isolated from two species of the genus Haageocereus (H. tenuis and H. pseudomelanostele) to be applied in studies of genetic diversity and population structure. ? Methods and Results: Five loci were employed in a preliminary study of genetic diversity and population differentiation in two rare (H. tenuis and H. repens) and two widespread (H. acranthus and H. pseudomelanostele) species, yielding between one and 44 alleles per locus. All five loci were polymorphic, with overall levels of observed and expected heterozygosities ranging from 0.478 to 0.871 and from 0.564 to 0.956, respectively. Three additional loci were scored in H. pseudomelanostele. These eight plus the remaining 11 loci were amplified from putative parents of three hybrids involving Haageocereus and Espostoa. ? Conclusions: These markers will facilitate analysis of genetic diversity, hybridization, and population differentiation throughout Haageocereus and Espostoa.     

NMR studies on domain diffusion and alignment in modular GB1 repeats

Modular proteins contain individual domains that are often connected by flexible, unstructured linkers. Using a model system based on the GB1 domain, we constructed tandem repeat proteins and investigated the rotational diffusion and long-range angular ordering behavior of individual domains by measuring NMR relaxation parameters and residual dipolar couplings. Although they display almost identical protein-solvent interfaces, each domain exhibits distinct rotational diffusion and alignment properties. The diffusion tensor anisotropy of the N-terminal domain (NTD) is D_‖/D_⊥ = 1.5-1.6, similar to that of single-GB1 domains (D_‖/D_⊥ = 1.6-1.7), whereas the value for the C-terminal domain (CTD) is D_‖/D_⊥ = 2.0-2.2. In addition, the two domains have different rotational correlation times. These effects are observed for linkers of three to 24 residues, irrespective of linker length. The NTD and CTD also differ in their degree of magnetic alignment, even with a flexible linker of 18 residues, exhibiting D_a values of 7.7 Hz and 9.7 Hz, respectively. Our results suggest that diffusion differences and long-range influences may persist in modular protein systems, even for systems that have highly flexible linkers and exhibit no domain-domain or domain-linker interactions.     

Evaluation of alternative technical markers for the pelvic coordinate system

In this study, we evaluated alternative technical markers for the motion analysis of the pelvic segment. Thirteen subjects walked eight times while tri-dimensional kinematics were recorded for one stride of each trial. Five marker sets were evaluated, and we compared the tilt, obliquity, and rotation angles of the pelvis segment: (1) standard: markers at the anterior and posterior superior iliac spines (ASIS and PSIS); (2) markers at the PSIS and at the hip joint centers, HJCs (estimated by a functional method and described with clusters of markers at the thighs); (3) markers at the PSIS and HJCs (estimated by a predictive method and described with clusters of markers at the thighs); (4) markers at the PSIS and HJCs (estimated by a predictive method and described with skin-mounted markers at the thighs based on the Helen-Hayes marker set); (5) markers at the PSIS and at the iliac spines. Concerning the pelvic angles, evaluation of the alternative technical marker sets evinced that all marker sets demonstrated similar precision across trials (about 1°) but different accuracies (ranging from 1° to 3°) in comparison to the standard marker set. We suggest that all the investigated marker sets are reliable alternatives to the standard pelvic marker set.     

Cytogenetic evidence for de novo synthesis of rRNA and involvement of nucleolar material in the organization of cell structures during spermiogenesis of Chariesterus armatus (Heteroptera, Coreidae)

The nucleolar material of Chariesterus armatus was analyzed during spermiogenesis in cell preparations impregnated with silver nitrate. Nucleolar corpuscles were observed in spermatids at the beginning of the process, showing that this organoid is also maintained after meiosis. In addition, nucleoli were seen in the round spermatids connected to the X-chromosome (bearer of the nucleolar organizer in C. armatus), indicating de novo synthesis of nucleolar material. This differs from the reorganization of ribosomal granules, transported from meiotic spermatocytes to round spermatids, where they would support protein synthesis, which is reported for other species. We also observed connections of nucleolar corpuscles to the nuclear membrane regions where the tail and the acrosome will be formed, suggesting close involvement of the nucleolar material in the formation of these structures. In addition to the nucleolar bodies, we detected silver-positive structures, which will require new approaches to clarify their role. One of these structures, observed in the cytoplasm, appears to correspond to the chromatoid body, which has been found in several organisms, but is still poorly understood; another is a complex structure to which the tail appears to be connected. We conclude that C. armatus is an appropriate model for understanding not only the synthesis of rRNA in the spermiogenesis, but also the functional meaning of the close relationship of nucleolar material with other structures during this process.     

Jiraiya attenuates BMP signaling by interfering with type II BMP receptors in neuroectodermal patterning

During embryogenesis, bone morphogenetic protein (BMP) signaling needs to be finely tuned in a locally restricted manner. Here, we report a cell-intrinsic mode of BMP response control executed by the membrane protein Jiraiya. In the Xenopus embryo, zygotic Jiraiya, expressed exclusively in the neuroectoderm, is essential and sufficient for limiting dorsal neural development, which is dependent on BMP signals. In animal cap assays, Jiraiya selectively and cell-autonomously inhibits BMP signaling, while Jiraiya's knockdown enhances the signaling. In the cell, Jiraiya selectively forms a complex with type II BMP receptor (BMPRII) and downregulates the cell surface localization of functional BMPRII. This functional interaction with Jiraiya depends on the unique tail domain of BMPRII, and, in particular, the conserved EVNNNG motif, the function of which has been unknown. Thus, Jiraiya represents a cell-intrinsic cutoff mechanism for dynamic responsiveness to BMP signals via subtype-selective receptor control.     

Cathepsin L Inhibition Prevents Murine Autoimmune Diabetes via Suppression of CD8+ T Cell Activity

Background

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice.

Methods and Findings

Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8⁺ T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8⁺ T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8⁺ T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice.

Conclusions

Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8⁺ T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strategy for autoimmune diabetes.     

Nucleobase catalysis in ribozyme mechanism

RNA performs a wide range of functions in biology including catalysis of chemical reactions. A major goal in the field of ribozyme chemical biology is to understand these functions in molecular terms. There is increasing evidence that ribozymes can use their nucleobases directly in chemical catalysis in a variety of ways. These include hydrogen bonding to the transition state, stabilizing charge development, and transferring protons as general acid-base catalysts. This article highlights recent kinetic, structural, single molecule, and synthetic approaches that have been used to probe the roles of ribozyme nucleobases in phosphodiester bond cleavage.     

Functional plasticity and catalytic efficiency in plant and bacterial ferredoxin-NADP(H) reductases

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. Two great families of FAD-containing proteins displaying FNR activity have evolved from different and independent origins. The enzymes present in mitochondria and some bacterial genera are members of the structural superfamily of disulfide oxidoreductases whose prototype is glutathione reductase. A second group, comprising the FNRs from plastids and most eubacteria, constitutes a unique family, the plant-type FNRs, totally unrelated in sequence with the former. The two-domain structure of the plant family of FNR also provides the basic scaffold for an extended superfamily of electron transfer flavoproteins. In this article we compare FNR flavoenzymes from very different origins and describe how the natural history of these reductases shaped structure, flavin conformation and catalytic activity to face the very different metabolic demands they have to deal with in their hosts. We show that plant-type FNRs can be classified into a plastidic class, characterised by extended FAD conformation and high catalytic efficiency, and a bacterial class displaying a folded FAD molecule and low turnover rates. Sequence alignments supported this classification, providing a criterion to predict the structural and biochemical properties of newly identified members of the family.     

Unique Distribution of <Emphasis Type="Italic">GAL</Emphasis> Genes on Chromosome XI in the Yeast <Emphasis Type="Italic">Saccharomyces naganishii</Emphasis>

A region of DNA extending from GAL7 to GAL1 was cloned in the yeast Saccharomyces naganishii. Sequence analysis revealed that GAL7 and GAL1 are separated by approximately 2 kbp and share a common promoter region. Although GAL7, GAL10, and GAL1 are clustered in this order in previously studied hemiascomycetous yeasts, GAL10 was not found between GAL7 and GAL1 in S. naganishii. Southern blotting of S. naganishii chromosomal DNA showed that both the GAL7–GAL1 region and GAL10 are located on chromosome XI, but that GAL10 is located more than 10 kbp away from GAL7–GAL1. Thus, S. naganishii and S. cerevisiae, while related phylogenetically, do not share the same orientation with respect to GAL genes. These data are highly relevant to studies of chromosomal evolution in yeast.