Extensive introgressive hybridization within the northern oriole group (Genus Icterus) revealed by three‐species isolation with migration analysis

Until recently, studies of divergence and gene flow among closely‐related taxa were generally limited to pairs of sister taxa. However, organisms frequently exchange genes with other non‐sister taxa. The “northern oriole” group within genus Icterus exemplifies this problem. This group involves the extensively studied hybrid zone between Baltimore oriole (Icterus galbula) and Bullock's oriole (I. bullockii), an alleged hybrid zone between I. bullockii and black‐backed oriole (I. abeillei), and likely mtDNA introgression between I. galbula and I. abeillei. Here, we examine the divergence population genetics of the entire northern oriole group using a multipopulation Isolation‐with‐Migration (IM) model. In accordance with Haldane's rule, nuclear loci introgress extensively beyond the I. galbula–I. bullockii hybrid zone, while mtDNA does not. We found no evidence of introgression between I. bullockii and I. abeillei or between I. galbula and I. abeillei when all three species were analyzed together in a three‐population model. However, traditional pairwise analysis suggested some nuclear introgression from I. abeillei into I. galbula, probably reflecting genetic contributions from I. bullockii unaccounted for in a two‐population model. Thus, only by including all members of this group in the analysis was it possible to rigorously estimate the level of gene flow among these three closely related species.     

The Cyanelles of Cyanophora Paradoxa

The cyanelles of Cyanophora paradoxa, plastids surrounded by a peptidoglycan wall, are considered as a surviving example for an early stage of plastid evolution from endosymbiotic cyanobacteria. We highlight the model character of the system by focusing on three aspects: “organelle wall” structure, plastid genome organization, and protein translocation.

The biosynthetic pathway for cyanelle peptidoglycan appears to be analogous to that in Escherichia coli. Also, the basic structure of this peculiar organelle wall corresponds to that of the E. coli sacculus, with one notable exception: the C-1 carboxyl group of the D-isoglutamyl residue is partially amidated with N-acetylputrescine. Cyanelles harbor on their completely sequenced 135.6-kb genome genes for approximately 150 polypeptides, many of which are nucleus encoded in higher plants. Nevertheless, there are striking parallels in genome organization between cyanelles (and other primitive plastids) and higher plant chloroplasts. The transit sequences of nucleus-encoded cyanelle preproteins resemble stroma targeting peptides of higher plant chloroplast precursors. Heterologous import of precursors from C. paradoxa into isolated pea chloroplasts is possible and vice versa. Cyanelles are considered to represent a very early, diverging branch of plastid evolution and are derived from the semiautonomous endosymbiont that had already abandoned about 90% of its genetic information but still retained its prokaryotic wall. Recent data on the molecular biology of cyanelles and rhodoplasts are consistent with the assumption of a primary endosymbiotic event that was not only monophyletic with respect to the cyanobacterial invader, but also singular.

Cyanophora paradoxa is the best-investigated member of the glaucocystophyceae, phototrophic protists containing cyanelles, that is, plastids stabilized by a peptidoglycan-containing envelope. The classification of this group, comprising only eight (mostly monotypic) genera, is also based on parallels in morphology and organization of the “host cells” (Kies, 1992). Recently, this was corroborated by 16S and 18S rRNA-based phylogenetic analysis (Helmchen et al., 1995; Bhattacharya et al, 1995). Apart from C. paradoxa, only Glaucocystis nostochinearum can be grown at a reasonable rate. Thus, biochemical and molecular genetic data are mostly available for C. paradoxa and more precisely for the isolate 555UTEX (Pringsheim) that is kept in the major culture collections of algae. Biochemical work done on C. paradoxa and the sequencing of individual cyanelle genes have been described in several recent reviews (Schenk, 1992; Löffelhardt and Bohnert, 1994a,b). Here we discuss three topics: the cyanelle wall, aspects deduced from the complete cyanelle genome sequence, and protein translocation into and within cyanelles.     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.     

Repeated cocaine administration changes the function and subcellular distribution of adenosine A1 receptor in the rat nucleus accumbens

Adenosine A1 receptor (A1) protein and mRNA is increased in the nucleus accumbens following repeated cocaine treatment. In spite of this protein up-regulation, A1 agonist-stimulated [35S]GTPgammaS binding was attenuated in accumbens homogenates of rats withdrawn for 3 weeks from 1 week of daily cocaine injections. Cellular subfractionation revealed that the discrepancy between total A1 protein and G protein coupling resulted from a smaller proportion of receptors in the plasma membrane. The decrease in functional receptor in the plasma membrane was further indicated by diminished formation of heteromeric receptor complex consisting of A1 and dopamine D1A receptors. To explore the functional significance of the altered distribution of A1 receptors, at 3 weeks after discontinuing repeated cocaine or saline, animals were injected with cocaine and 45 min later the subcellular distribution of A1 receptors quantified. Whereas a cocaine challenge in repeated saline-treated animals induced a marked increase in membrane localization of the A1 receptor, the relative distribution of receptors in repeated cocaine rats was not affected by acute cocaine. These data suggest that the sorting and recycling of A1 receptors is dysregulated in the nucleus accumbens as the consequence of repeated cocaine administration.     

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Zooplankton community structure along a pollution gradient at fine geographical scales in river ecosystems: The importance of species sorting over dispersal

The release of anthropogenic pollution into freshwater ecosystems has largely transformed biodiversity and its geographical distribution patterns globally. However, for many communities including ecologically crucial ones such as zooplankton, it is largely unknown how different communities respond to environmental pollution. Collectively, dispersal and species sorting are two competing processes in determining the structure and geographical distribution of zooplankton communities in running water ecosystems such as rivers. At fine geographical scales, dispersal is usually considered as the dominant factor; however, the relative role of species sorting has not been evaluated well, mainly because significant environmental gradients rarely exist along continuously flowing rivers. The Chaobai River in northern China represents a rare system, where a significant environmental gradient exists at fine scales. Here, we employed high‐throughput sequencing to characterize complex zooplankton communities collected from the Chaobai River, and tested the relative roles of dispersal and species sorting in determining zooplankton community structure along the pollution gradient. Our results showed distinct patterns of zooplankton communities along the environmental gradient, and chemical pollutant‐related factors such as total phosphorus and chlorophyll‐a were identified as the major drivers for the observed patterns. Further partial redundancy analyses showed that species sorting overrode the effect of dispersal to shape local zooplankton community structure. Thus, our results reject the dispersal hypothesis and support the concept that species sorting caused by local pollution can largely determine the zooplankton community structure when significant environmental gradients exist at fine geographical scales in highly polluted running water ecosystems.     

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Plant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.     

Five-in-One: Simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.     

The Golgi complex: a hub of the secretory pathway

The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance.