Synthesis of keyhole limpet hemocyanin by the rhogocytes of Megathura crenulata

Rhogocytes are morphologically distinct cells distributed throughout connective tissues of crustaceans and molluscs. Using light microscopy, rhogocytes of the vetigastropod Megathura crenulata were identified by their ovoid shape, and their cytoplasm filled with spherical inclusions which contained lysosomal enzymes, based on uptake of neutral red and staining with LysoTracker dye. Rhogocytes were most abundant in the digestive gland (2,824 rhogocytes/mm²), followed by the connective tissue layer surrounding the middle and posterior esophagus and intestine (1,431 rhogocytes/mm², 872 rhogocytes/mm², and 1,190 rhogocytes/mm², respectively), and were lowest in abundance in the foot (154 rhogocytes/mm²). At the transmission electron microscopy level, characteristic features of rhogocytes were inclusions showing a variety of electron densities, abundant vesicles, and rough endoplasmic reticulum in the cytoplasm, and regions of plasma membrane folded to produce slits connected by thin diaphragms. Although several functions have been proposed for gastropod rhogocytes, much attention has been focused on their possible role in the synthesis of the respiratory pigment hemocyanin. In M. crenulata, this molecule exists in several isoforms called keyhole limpet hemocyanin (KLH). One isoform, KLH1, is a large didecamer and has been used extensively in studies on vertebrate immunology and cancer therapy. We present four lines of evidence indicating rhogocytes in M. crenulata synthesize KLH1. First, at the transmission electron microscopy (TEM) level, dilated cisternae of RER containing material similar in size and shape to KLH were observed in rhogocytes examined throughout the year. Second, KLH1 mRNA was identified exclusively in tissue samples that contained rhogocytes; no mRNA for KLH1 was identified in samples containing only hemocytes. Third, immunoperoxidase staining with antibodies specific to KLH was localized only to rhogocytes. Fourth, in situ hybridization with a probe specific for M. crenulata KLH1 demonstrated KLH1‐specific mRNA was present only in rhogocytes. Identification of the cells responsible for the synthesis of KLH is important because of the clinical significance of this molecule.     

Recognition of O6-benzyl-2′-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction

The 2′-deoxynucleoside containing the synthetic base 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-perimidin-2(3H)-one] (dPer) recognizes in DNA the O⁶-benzyl-2′-deoxyguanosine nucleoside (O⁶-Bn-dG), formed by exposure to N-benzylmethylnitrosamine. Herein, we show how dPer distinguishes between O⁶-Bn-dG and dG in DNA. The structure of the modified Dickerson–Drew dodecamer (DDD) in which guanine at position G⁴ has been replaced by O⁶-Bn-dG and cytosine C⁹ has been replaced with dPer to form the modified O⁶-Bn-dG:dPer (DDD-XY) duplex [5′-d(C¹G²C³X⁴A⁵A⁶T⁷T⁸Y⁹G¹⁰C¹¹G¹²)-3′]₂ (X = O⁶-Bn-dG, Y = dPer) reveals that dPer intercalates into the duplex and adopts the syn conformation about the glycosyl bond. This provides a binding pocket that allows the benzyl group of O⁶-Bn-dG to intercalate between Per and thymine of the 3′-neighbor A:T base pair. Nuclear magnetic resonance data suggest that a similar intercalative recognition mechanism applies in this sequence in solution. However, in solution, the benzyl ring of O⁶-Bn-dG undergoes rotation on the nuclear magnetic resonance time scale. In contrast, the structure of the modified DDD in which cytosine at position C⁹ is replaced with dPer to form the dG:dPer (DDD-GY) [5′-d(C¹G²C³G⁴A⁵A⁶T⁷T⁸Y⁹G¹⁰C¹¹G¹²)-3′]₂ duplex (Y = dPer) reveals that dPer adopts the anti conformation about the glycosyl bond and forms a less stable wobble pairing interaction with guanine.     

Vegetation trajectories of restored agricultural wetlands following sediment removal

Recognition of wetland ecosystem services has led to substantial investment in wetland restoration in recent decades. Wetland restorations can be designed to meet numerous goals, among which reestablishing a diverse native wetland plant community is a common aim. In agricultural areas, where previously drained wetland basins can fill with eroded sediment from the surrounding landscape, restoration often includes excavation to expose buried seed banks. The extent to which excavation improves the diversity of wetland plant communities is unclear, particularly in terms of longer‐term outcomes. We examined plant species diversity and community composition in 24 restored agricultural wetlands across west‐central Minnesota, U.S.A. In all study wetlands, hydrology was restored by removing subsurface drainage and plugging drainage ditches, thus reestablishing groundwater connectivity and hydroperiod (“business as usual” treatment). In half of the wetlands, accumulated sediment was removed from the basin and redeposited on the surrounding landscape (“excavated” treatment). Initially, sediment removal significantly decreased invasive species cover, particularly of hybrid cattail (Typha × glauca) and reed canary grass (Phalaris arundinacea), and increased community diversity and evenness. Over time, the effects of sediment removal diminished, and eventually disappeared by approximately 6 years after restoration. While our results demonstrate that sediment removal improves initial restoration outcomes for plant communities, longer‐term benefits require sustained management, such as invasive species control or resetting of basins through additional excavation.     

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post-translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC-MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism.     

Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1–depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.     

Methane-based symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan

Abstract. Bathymodiolus platifrons , a chemosynthetic mussel from cold seeps off Japan, relies for its nutrition on the productivity of methylotrophic or methanotrophic endosymbionts. High densities of bacterial symbionts appearing to be type I methanotrophs were observed in transmission electron micrographs of gill tissues. Methanol dehydrogenase activity in gill tissue from a single individual was positive compared to non-methanotrophic control samples, indicating a high potential for methanotrophy. Stable isotopic ratios of carbon in symbiont-containing gill tissue, as well as host tissues, were extremely depleted in ¹³C, and similar to values reported for other methanotrophic species. TEMs of gill tissue showing symbionts in various stages of digestion support the hypothesis that carbon transfer from symbionts to B. platifrons occurs through intracellular digestion of the symbionts. Discovery of methane- or methanolbased symbioses in B. platifrons from cold seeps in Sagami Bay extends the range of such symbioses to include cold seeps and hydrothermal vents, and supports the idea that environmental methane levels control the distribution of these symbioses.