PKD controls mitotic Golgi complex fragmentation through a Raf–MEK1 pathway

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to–cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1–dependent manner, a process required for entry of the cells into mitosis.     

Plant-insect communities and predator-prey ratios in field margin strips,adjacent crop fields,and fallows

The management of field margin strips for the enhancement of biodiversity of plant-insect communities and natural-enemy populations was studied on experimental farms near Göttingen (Germany). Young and old, sown and naturally developed field margin strips were compared and differences to large fallows established. The five types of field margin strips (around cereal fields) were: (1, 2) 1- or 6-year-old naturally developed strips, (3) strips sown with a Phacelia mixture, (4) strips sown with a mixture of 19 wild flower species, and (5) strips sown with winter wheat or oat as a control. The naturally developed vegetation of the field margin strips was dominated by aggressive weeds, presumably due to the intensive farming practices and the fertile soils. Cirsium arvense populations decreased, while Elymus repens populations increased with age of habitat. Sowings were suitable to suppress these aggressive weeds. Potted plants of mugwort (Artemisia vulgaris) and red clover (Trifolium pratense) were exposed in the field margin strips to study arthropod colonization of these experimentally standardized plant patches. Arthropod species richness did not differ between field margin types, reflecting the overall similarity in floristic diversity, but sprayed and strip-free edges of cereal fields had a reduced diversity. Dispersal of insect populations of red clover into the cereal fields decreased with increasing distance, but benefited from adjacent field margin strips. Populations of predators (mainly spiders) as well as predator-prey ratios were significantly larger in 6-year-old than in 1-year-old strips emphasizing the importance of habitat age for natural enemies and possible biological control. Predator-prey ratios were also higher on old than young fallows. Large fallows had greater predator-prey ratios than small field margin strips emphasizing the trophic-level hypothesis of island biogeography in that the relative importance of natural enemies increased with habitat area. Insect species richness was only marginally influenced by area and not by age. As species richness of predators did not increase with area and age, species diversity and the possible biological-control function did not covary.     

Vertical migration patterns in phytoplankton of mixed species composition

The vertical distribution and migration of phytoplankton composed of seven organisms in a small eutrophic pool is described. Vertical migration during a 24 h period is shown to be exhibited by Pandorina morum, Mallomonas tonsurata, Chroomonas pusilla (Rhodomonas) and Ochromonas sp. in the epilimnion and Cryptomonas rufescens in the surface of the hypolimnion. The degree of horizontal variation in distribution of the organisms is discussed and found to be greatest for the microflagellates. Changes in vertical distribution are shown to be significant and result from active movement of the cells rather than from either water movement or the combined variation associated with patchiness in distribution and counting errors.

Differences between the behaviour patterns of the different algae are evident and may be used to explain Hutchinson's “paradox of the plankton”.     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Rational design and synthesis of novel nucleotide anti-Giardia agents

Design and synthesis of a novel nucleotide anti-Giardia agent that is micromolar inhibitor of Giardia trophozoite growth in culture is described.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.     

Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs

Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.     

A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell

Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.