Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.     

Whole Cell Screen for Inhibitors of pH Homeostasis in Mycobacterium tuberculosis

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pH_IB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pH_IB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.     

Ultrastructural study of embryonic and early adult germ cells,and their support cells,in both sexes of Xiphophorus (Teleostei:Poeciliidae)

The ultrastructure of the early spermatogonia in mature testes of the platyfish, Xiphophorus maculatus, was compared to that of oogonia in mature ovaries of X. maculatus and the related X. nigrensis. Both cell types were very similar and, characterized as being large, oval to round cells containing large, central nuclei with prominent nucleoli. Abundant mitochondria with sparse transverse cristae were located at one pole or around the nucleus. Annulate lamellae and electron-dense granular material (nuage) were present. Other organelles were not prominent. A female that had received a testis graft had testicular tissue containing mature spermatozoa within the ovary, indicating that cells were present that could develop along the male line. Special crosses were carried out to obtain all-male embryos of X. maculatus and all-female embryos of X. nigrensis. The ultrastructure of the germ cells in all embryonic gonads was similar to that of the adult cells. These results suggest the presence of sexually undifferentiated germ cells in the adult gonads of both sexes. The support cells investing all of these germ cells were also similar structurally and appeared to be undifferentiated.     

Comparative immunochemical and structural similarities of five stress proteins from various tissue types

1. Incorporation of radioactive phosphate or leucine by stress proteins from rodent lymphoma, submaxillary and liver nuclei were observed in two-dimensional gels following chemical and environmental stress. 2. The stress proteins were isolated from second-dimension gels and their similarities compared. Mol. wt determinations, immunochemical blotting and protease V8 peptide mapping confirmed the identical nature of the stress proteins possessing identical Mr, but from diverse tissue types. 3. These data imply that highly similar stress proteins exist in diverse tissues, are conserved during evolution, and possess some elemental and essential function.     

Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1

The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P(1) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.     

Endogenous testosterone levels, mental rotation performance, and constituent abilities in middle-to-older aged men

Evidence from both human and animal studies suggests that gonadal steroids, such as testosterone, exert activational effects on adult spatial behavior. Endogenous testosterone levels decline gradually but variably as men age; it remains to be shown whether these decreases are associated with age-related declines in visuo-spatial performance or constituent abilities indicative of generalized age-related cognitive decline. Ninety-six healthy, community dwelling men aged between 38 and 69 years completed the Vandenberg and Kuse Mental Rotation Test (MRT) together with a battery of tests including processing speed, executive function, perceptual discrimination, working memory, and reaction time measures. Significant main effects of tertiles of calculated free testosterone levels (cEFT) were found on composite measures of processing speed, executive function, and perceptual discrimination ability in a subset of men aged over 50 years in age and crystallized intelligence controlled analyses; higher cEFT levels were associated with poorer performance. Hierarchical multiple regression and path analyses on the whole data set showed that cEFT levels negatively moderated processing speed performance, which in turn predicted both working memory and MRT performance with aging. Together these data suggest that age-related declines in endogenous testosterone levels in healthy middle-to-older aged men are not associated with generalized age-related cognitive decline.     

A multi-scale test for dispersal filters in an island plant community

Constraints on plant distributions resulting from seed limitation (i.e. dispersal filters) were evaluated on two scales of ecological organization on islands off the coast of British Columbia, Canada. First, island plant communities were separated into groups based on fruit morphology, and patterns in species diversity were compared between fruit‐type groups. Second, abundance patterns in several common fleshy‐fruited, woody angiosperm species were compared to species‐specific patterns in seed dispersal by birds. Results from community‐level analyses showed evidence for dispersal filters. Dry‐fruited species were rare on islands, despite being common on the mainland. Island plant communities were instead dominated by fleshy‐fruited species. Patterns in seed dispersal were consistent with differences in diversity, as birds dispersed thousands of fleshy‐fruited seeds out to islands, while dry fruited species showed no evidence of mainland‐island dispersal. Results from population‐level analyses showed no evidence for dispersal filters. Population sizes of common fleshy‐fruited species were unrelated to island isolation, as were rates of seed dispersal. Therefore, island isolation distances were not large enough to impose constraints on species’ distributions resulting from seed limitation. Rates of seed dispersal were also unrelated to island area. However, several species increased in abundance with island area, indicating post‐dispersal processes also help to shape species distributions. Overall results suggest that seed dispersal processes play an important role in determining the diversity and distribution of plants on islands. At the community‐level, dry‐fruited species were seed limited and island communities were instead dominated by fleshy‐fruited species. At the population‐level, common fleshy‐fruited species were not seed limited and showed few differences in distribution among islands. Therefore, although evidence for dispersal filters was observed, their effects on plant distributions were scale‐dependent.     

Association of the signaling adaptor FRS2 with fibroblast growth factor receptor 1 (Fgfr1) is mediated by alternative splicing of the juxtamembrane domain.

Fibroblast growth factor receptors (FGFRs) are a family of transmembrane tyrosine kinases involved in signaling via interactions with the family of fibroblast growth factors. Alternative splicing of the juxtamembrane region of FGFR1-3 leads to the inclusion or exclusion of two amino acids, valine and threonine, the VT site. The presence or absence of VT (VT+ or VT-, respectively) affects the signaling potential of the receptor. The VT+ receptor isoform is required for Erk2 phosphorylation, a component of the mitogen-activated protein kinase signaling pathway. FRS2 is an adaptor protein that links FGFRs to the mitogen-activated protein kinase signaling pathway. FRS2 interacts with a region of the juxtamembrane domain of FGFR1 that includes the alternatively spliced VT site. We investigated the interaction of FRS2 with murine Fgfr1 juxtamembrane domain. We showed the alternatively spliced VT motif, at the juxtamembrane domain of Fgfr1 is required for FRS2 interaction with Fgfr1. Activation of signaling pathways from FRS2 is likely to be regulated by controlling the Fgfr1/FRS2 interaction through alternative splicing of the VT motif of Fgfr1.