Cyclic AMP relay and cyclic AMP-induced cyclic GMP accumulation during development of Dictyostelium discoideum

Abstract Cyclic AMP-induced cAMP and cGMP responses during development of Dictyostelium discoideum were investigated. The cAMP-induced cGMP response is maximal when aggregation is in full progress, and then decreases to about 10% of the maximal level during further multicellular development. The cAMP response increases upon starvation, reaches its maximum at the onset of aggregation, and then decreases to about 8% of the maximum level. The dynamics of the post-aggregative cAMP response are in qualitative agreement with the dynamics of the cAMP relay response in aggregation-competent cells.     

ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal–ventral patterning during early embryogenesis in Xenopus.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Genetics of Azospirillum brasilense with respect to ammonium transport,sugar uptake,and chemotaxis

This paper describes molecular aspects of Azospirillum-plant root association with respect to nitrogen flux and carbon utilization. In the first part, biochemical and genetic data are reported on the transport of ammonium and methylammonium in A. brasilense cells. Ammonium excreting A. brasilense mutants reported so far appear to result from alterations in genes encoding for enzymes involved in ammonium assimilation. Solid genetic evidence is given on the occurrence of a postulated ammonium transporter in A. brasilense. In the second part, biochemical and genetic evidence is likewise given for the occurrence of a high-affinity uptake system for D-galactose in A. brasilense. A sugar- binding protein that is part of this uptake system is required for chemotaxis of A. brasilense towards particular sugars, including D-galactose.     

The dcr gene family of Desulfovibrio: Implications from the sequence of dcrH and phylogenetic comparison with other mcp genes

Desulfovibrio vulgaris Hildenborough contains a family of genes for methyl-accepting chemotaxis proteins (MCPs). Here we report the complete sequence of the gene for Desulfovibrio chemoreceptor H (dcrH). The deduced amino acid sequence of DcrH protein, which has an enlarged N-terminal, ligand binding domain, indicates a structure similar to that of other MCPs. Comparison of the sequences for DcrA, determined earlier, and DcrH indicated that similarity is essentially limited to the C-terminal excitation region. The dcr gene family differs, in this respect, from mcp gene families in other eubacteria (e.g. Escherichia coli and Bacillus subtilis), where MCPs share significant homology throughout their C-terminal signal transduction domains. This may point to an ancient evolutionary origin of the dcr gene family, which is widely distributed throughout the genus Desulfovibrio. The evolutionary origin of mcp genes was traced by comparing nucleotide sequences for the excitation region that is common to all MCPs. Phylogenetic analysis of sequences for thirty mcp genes from nine eubacterial and one archaebacterial species suggested that multiplication of mcp genes has occurred at least twice since the eubacteria diverged from the archaebacteria.Nucleotide accession number: The nucleotide sequence reported in this paper has been entered into GenBank under accession number U30319. Phone: 403-220-6388. Fax: 403-289-9311. Electronic mail address: voordouw@acs.ucalgary.ca.     

Dose-related upwind anemotaxis and movement up odour gradients in still air in the presence of methyl eugenol by the wild tobacco fly, Bactrocera cacuminata

The behaviour of Bactrocera cacuminata (Hering) in wind varied according to the concentration of methyl eugenol (0, 95, 327 and 500 μg m^?3, respectively). General locomotor activity (as measured by mean distance moved in 5 min, regardless of direction) was not significantly different in the first two treatments but was significantly lower in the others. Most flies in the fourth treatment did not move more than one body length. In the first two treatments, the rate and pattern of movement of most flies was basically similar, with walking in tortuous paths interspersed with short flights and usually no obvious bias in direction. However, 32% of flies in the second treatment did move in a biased direction, achieving upwind anemotaxis of at least 400 mm, but only 2–8% did so in the other conditions. Flies moved up a concentration gradient to a source of methyl eugenol in still air when released at a distance of 100, 150 or 200 mm. With one exception, no more than 40% did this within 3 min of release (whether or not the olfactory stimulus was augmented by a visual one). However, 77% responded when released 100 mm from a combined olfactory and visual stimulus. Visual augmentation of an olfactory stimulus may also be responsible for far fewer flies flying out of the vicinity at distances up to 150 mm, but not 200 mm.     

微生物对水环境污染物的趋化性研究进展

随着人类活动的干扰以及社会工业化进程的加剧,水环境污染现象日益严峻。微生物是水环境污染治理和修复的主要驱动者。越来越多的研究发现,水环境中污染物的去除速率与微生物的趋化功能密切相关。综述了近几年有关微生物对水环境中无机盐、溶解性有机物和重金属等的趋化特性研究进展,讨论并展望了基于趋化功能微生物精准调控的水环境污染强化治理技术的应用前景及主要问题。     

D-Glucose but Not Insulin Reduces N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-Induced Shape Changes in Suspended Human Neutrophils

The effects of glucose (5–25 mM) and insulin concentration (40–320 U/ml) on the cell shape of neutrophil granulocytes from healthy humans were studied. Both non-activated and N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-activated neutrophils in suspension were used as a model for initial chemotactic activation of neutrophil locomotion. D-glucose, but not the non-metabolizable analogue 3-O-methyl-D-glucose, dose-dependently reduced the fMet-Leu-Phe-induced (10^–8M) neutrophil elongation. Insulin, either alone or in combination with 25 mM D-glucose, was without effect on the fMet-Leu-Phe-induced neutrophil elongation. Furthermore, the inhibitory effect of D-glucose was observed already after 1 min of exposure to D-glucose and fMet-Leu-Phe. D-glucose diminished the fraction of neutrophils with elongated locomotor shape by changing it into an irregular cell shape, suggesting that at least part of the D-glucose effect could be associated with mechanisms determining the typical locomotor shape. The present results suggest that D-glucose through its metabolism, but without the involvement of insulin, reduces chemotactically induced elongation to a locomotor neutrophil shape, and thus neutrophil motility, and that this effect of glucose appears prior to adhesion. This glucose-induced inhibition of the neutrophil chemotactic response may be involved in the neutrophil deficiency seen in diabetes mellitus.     

Synthesis,conformation, and biological activity of two fMLP-OMe analogues containing the new 2-[2′-(methylthio) ethyl] methionine residue

The new C^α-tetrasubstituted α-amino acid residue 2-[2′-(methylthio) ethyl]methionine (Dmt) has been introduced into the reference chemotactic tripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in place of the leucine or methionine, respectively. The biological activity of the new analogues [Dmt²] fMLP-OMe (2) and [Dmt¹] fMLP-OMe (3) has been determined; whereas 2 is active toward human neutrophils, stimulating directed migration, superoxide anion generation, and lysozyme release, 3 results practically inactive in all tested assays. A conformational analysis on 2 and 3 has been performed in solution by using ir absorption and ¹H-nmr. The conformation of 2 was also examined in the crystal by x-ray diffraction methods. Both 2 and 3 adopt fully extended conformation in correspondence with the Dmt residue. Biological and conformational results are discussed and compared with related previously studied models. © 1997 John Wiley & Sons, Inc. Biopoly 42: 415–426, 1997     

Production,characterization, and assessment of a stable analog of the response regulator CheY‐phosphate from Thermotoga maritima

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY‐phosphate have been challenging due to the rapid hydrolysis of the aspartyl‐phosphate in vitro. An inert analog of Tm CheY‐phosphate, phosphono‐CheY, was synthesized by chemical modification and purified by cation‐exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono‐CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono‐CheY bound a peptide derived from FliM, a native partner of CheY‐phosphate, with a dissociation constant of ～29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono‐CheY is a useful and unique analog of Tm CheY‐phosphate.