On the Ultrastructure and Function of Rhogocytes from the Pond Snail Lymnaea stagnalis

Rhogocytes, also termed “pore cells”, occur as solitary or clustered cells in the connective tissue of gastropod molluscs. Rhogocytes possess an enveloping lamina of extracellular matrix and enigmatic extracellular lacunae bridged by cytoplasmic bars that form 20 nm diaphragmatic slits likely to act as a molecular sieve. Recent papers highlight the embryogenesis and ultrastructure of these cells, and their role in heavy metal detoxification. Rhogocytes are the site of hemocyanin or hemoglobin biosynthesis in gastropods. Based on electron microscopy, we recently proposed a possible pathway of hemoglobin exocytosis through the slit apparatus, and provided molecular evidence of a common phylogenetic origin of molluscan rhogocytes, insect nephrocytes and vertebrate podocytes. However, the previously proposed secretion mode of the respiratory proteins into the hemolymph is still rather hypothetical, and the possible role of rhogocytes in detoxification requires additional data. Although our previous study on rhogocytes of the red-blooded (hemoglobin-containing) freshwater snail Biomphalaria glabrata provided much new information, a disadvantage was that the hemoglobin molecules were not unequivocally defined in the electron microscope. This made it difficult to trace the exocytosis pathway of this protein. Therefore, we have now performed a similar study on the rhogocytes of the blue-blooded (hemocyanin-containing) freshwater snail Lymnaea stagnalis. The intracellular hemocyanin could be identified in the electron microscope, either as individual molecules or as pseudo-crystalline arrays. Based on 3D-electron microscopy, and supplemented by in situ hybridization, immunocytochemistry and stress response experiments, we provide here additional details on the structure and hemocyanin biosynthesis of rhogocytes, and on their response in animals under cadmium and starvation stress. Moreover, we present an advanced model on the release of synthesized hemocyanin molecules through the slit apparatus into the hemolymph, and the uptake of much smaller particles such as cadmium ions from the hemolymph through the slit apparatus into the cytoplasm.     

Electron paramagnetic resonance study of radiation damage in photosynthetic reaction center crystals

Electron paramagnetic resonance (EPR) was used to simultaneously study radiation-induced cofactor reduction and damaging radical formation in single crystals of the bacterial reaction center (RC). Crystals of Fe-removed/Zn-replaced RC protein from Rhodobacter ( R.) sphaeroides R26 were irradiated with varied radiation doses at cryogenic temperature and analyzed for radiation-induced free radical formation and alteration of light-induced photosynthetic electron transfer activity using high-field (HF) D-band (130 GHz) and X-band (9.5 GHz) EPR spectroscopies. These analyses show that the formation of radiation-induced free radicals saturated at doses 1 order of magnitude smaller than the amount of radiation at which protein crystals lose their diffraction quality, while light-induced RC activity was found to be lost at radiation doses at least 1 order of magnitude lower than the dose at which radiation-induced radicals exhibited saturation. HF D-band EPR spectra provide direct evidence for radiation-induced reduction of the quinones and possibly other cofactors. These results demonstrate that substantial radiation damage is likely to have occurred during X-ray diffraction data collection used for photosynthetic RC structure determination. Thus, both radiation-induced loss of photochemical activity in RC crystals and reduction of the quinones are important factors that must be considered when correlating spectroscopic and crystallographic measurements of quinone site structures.     

Cerebral Cavernous Malformation 2 Protein Promotes Smad Ubiquitin Regulatory Factor 1-mediated RhoA Degradation in Endothelial Cells

Mutation of CCM2 predisposes individuals to cerebral cavernous malformations, vascular abnormalities that cause seizures and hemorrhagic stroke. CCM2 has been proposed to regulate the activity of RhoA for maintenance of vascular integrity. Herein, we define a novel mechanism where the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3) Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown of CCM2 have increased RhoA protein and display impaired directed cell migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to localize Smurf1 for RhoA degradation. These findings provide a molecular mechanism for the pathogenesis of cerebral cavernous malformations (CCM) resulting from loss of CCM2-mediated localization of Smurf1, which controls RhoA degradation required for maintenance of normal endothelial cell physiology.We previously characterized a scaffold-like protein named osmosensing scaffold for MEKK3 (OSM) for its ability to bind actin and localize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to hyperosmotic stress (1). Subsequently, the gene encoding OSM, CCM2, was found to be mutated in the human disease cerebral cavernous malformations (CCM)² (2). Cerebral cavernous malformations are vascular lesions of the central nervous system characterized as clusters of dilated, thin walled blood vessels. CCM lesions are fragile and prone to vascular leakiness and rupture, leading to hemorrhages that cause seizure and stroke (3, 4).Recently, CCM2 knockdown endothelial cells were shown to have increased activation of RhoA (5), although the mechanism was not defined. Herein, we demonstrate a molecular mechanism for activation of this pathway. Through a novel CCM2 PTB domain interaction with the Smurf1 homologous to the E6-AP C terminus (HECT) domain, we now show that CCM2 binds the E3 ligase Smurf1 for the control of RhoA degradation.     

Whole mount nuclear fluorescent imaging: Convenient documentation of embryo morphology

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. genesis 2012. © 2012 Wiley Periodicals, Inc.     

Nup53 is required for nuclear envelope and nuclear pore complex assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.     

Delineating genetic relationships among the Maya

By 250 AD, the Classic Maya had become the most advanced civilization within the New World, possessing the only well-developed hieroglyphic writing system of the time and an advanced knowledge of mathematics, astronomy and architecture. Though only ruins of the empire remain, 7.5 million Mayan descendants still occupy areas of Mexico, Guatemala, Belize, El Salvador, and Honduras. Although they inhabit distant and distinct territories, speak more than 28 languages, and have been historically divided by warfare and a city-state-like political system, and they share characteristics such as rituals, artistic, architectural motifs that distinguish them as unequivocally Maya. This study was undertaken to determine whether these similarities among Mayan communities mirror genetic affinities or are merely a reflection of their common culture. Four Mayan populations were investigated (i.e., the K'iche and Kakchikel from Guatemala and the Campeche and Yucatan from Mexico) and compared with previously published populations across 15 autosomal STR loci. As a whole, the Maya emerge as a distinct group within Mesoamerica, indicating that they are more similar to each other than to other Mesoamerican groups. The data suggest that although geographic and political boundaries existed among Mayan communities, genetic exchanges between the different Mayan groups have occurred, supporting theories of extensive trading throughout the empire.     

Genetics of species differences in the wild annual sunflowers, Helianthus annuus and H. petiolaris

Much of our knowledge of speciation genetics stems from quantitative trait locus (QTL) studies. However, interpretations of the size and distribution of QTL underlying species differences are complicated by differences in the way QTL magnitudes are estimated. Also, many studies fail to exploit information about QTL directions or to compare inter- and intraspecific QTL variation. Here, we comprehensively analyze an extensive QTL data set for an interspecific backcross between two wild annual sunflowers, Helianthus annuus and H. petiolaris, interpret different estimates of QTL magnitudes, identify trait groups that have diverged through selection, and compare inter- and intraspecific QTL magnitudes. Our results indicate that even minor QTL (in terms of backcross variance) may be surprisingly large compared to levels of standing variation in the parental species or phenotypic differences between them. Morphological traits, particularly flower morphology, were more strongly or consistently selected than life history or physiological traits. Also, intraspecific QTL were generally smaller than interspecific ones, consistent with the prediction that larger QTL are more likely to spread to fixation across a subdivided population. Our results inform the genetics of species differences in Helianthus and suggest an approach for the simultaneous mapping of inter- and intraspecific QTL.     

COMBINING NEW TECHNOLOGIES FOR DETERMINATION OF PHYTOPLANKTON COMMUNITY STRUCTURE IN THE NORTHERN GULF OF MEXICO 1

In situ analysis of phytoplankton community structure was determined at five stations along the Texas Gulf coast using two instruments, the Fluoroprobe and FlowCAM. Results were compared with traditional methods to determine community structure (pigment analysis and microscopy). Diatoms and small nanoplankton (most likely haptophytes) dominated the phytoplankton community at all stations. Estimated chl concentrations for diatoms+dinoflagellates obtained via the Fluoroprobe were not significantly different for three of the five stations sampled when compared with HPLC‐chemical taxonomy analysis, whereas the concentrations of green algal and cryptophyte chl were overestimated. The FlowCAM estimates of overall nanoplankton and microplankton cell abundance were not significantly different when compared with epifluorescence microscopy, and recorded images of phytoplankton cells provided a representative population of the phytoplankton community at each station. The Fluoroprobe and FlowCAM, when used in tandem, are potentially capable of determining the general characteristics of phytoplankton community structure in situ and could be an important addition to biological observing systems in the coastal ocean.     

Primate Conservation Biology

Primate Conservation Biology. Guy Cowlishaw and Robin Dunbar. Chicago: University of Chicago Press, 2000. 498 pp.