Studies on cellular adhesion of Xenopus laevis melanophores: modulation of cell-cell and cell-substratum adhesion in vitro by endogenous Xenopus galactoside-binding lectin

We have investigated cell-cell and cell-substratum adhesion of Xenopus laevis neural crest cells at various stages of melanophore differentiation. Single-cell suspensions were obtained by trypsinization and aggregated in a cell-cell adhesion assay. Unpigmented cells did not adhere while the rate of adhesion of melanophores correlated with the degree of melanization. Melanophore cell-cell adhesion decreased significantly in the presence of beta-galactosidase, which suggests that cell-surface galactose is involved. Beta-galactoside-binding lectin has been isolated and purified from embryos at the stage of neural crest migration. When added to aggregating cells smaller, looser clusters formed compared to controls. When lectin was added to cells in stationary culture to test cell-substratum adhesion, melanophores spread more smoothly and formed more regular spacing patterns. These results suggest that this lectin can modulate receptors used in cell-cell and cell-substratum adhesion of melanophores.     

The effects of various nutritional supplements on the growth,migration and differentiation ofxenopus laevis neural crest cells in vitro

Summary This study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested for cell migration and adhesion. This work was supported by establishment and major equipment grants from the Alberta Heritage Foundation for Medical Research to N. C. M. Nadine C. Milos is a Heritage Medical Research Scholar of the Alberta Heritage Foundation for Medical Research.     

Spike discharges of skeletomotor neurons during random noise modulated transmembrane current stimulation and muscle stretch

 Spike discharges of skeletomotor neurons innervating triceps surae muscles elicited by white noise modulated transmembrane current stimulation and muscle stretch were studied in decerebrated cats. The white noise modulated current intensity ranged from 4.3 to 63.2 nA peak-to-peak, while muscle stretches ranged from 100 μm to 4.26 mm peak-to-peak. The neuronal responses were studied by averaging the muscle length records centered at the skeletomotor action potentials (peri-spike average, PSA) and by Wiener analysis. Skeletomotor spikes appeared after a sharp peak in PSA of the injected current, preceded by a longer-lasting smaller wavelet of either depolarizing or hyperpolarizing direction. The PSA amplitude was not related to the injected current amplitude nor showed any differences related to the motor unit type. The PSA amplitudes were virtually independent of the stretching amplitude σ, after an initial increase with stretching amplitudes in the range of 15–40 μm (S.D.), or 100–270 μm peak-to-peak.Analyses of cross-spectra indicated a small or absent increase in gain with frequency in response to injected current, but about 20 dB/decade in the range 10–100 Hz in response to muscle stretch. The peaks of both Wiener kernels in response to current injection appear to decrease with the amplitude of injected current, but this decrease was not statistically significant. The narrow first-order kernels suggest that the transfer function between the current input and spike discharge is lowpass with a wide passband, i.e. there is very little change in dynamics. The values of the second-order kernels appear to be nonzero only along the main diagonal. This is characteristic of a simple Hammerstein type cascade, i.e. a zero memory nonlinearity followed by a linear system. Small values of second-order kernels away from the origin and narrow first-order kernels suggest that the linear cascade contributes very little to the overall dynamic response.In contrast to Wiener kernels found in response to current injection, the Wiener kernels in response to stretch showed a decreasing trend with stretch amplitude. The size of the second-order kernels decreased to a somewhat larger extent with input amplitude than that of the first-order kernels, indicating an amplitude-dependent nonlinearity. Overall, the transformation between length and spike output was described as an LNNL cascade with second-order nonlinearities. Received: 1 April 1993/Accepted in revised form: 24 March 1994     

Preservation and computer-aided microscopic analysis of planktonic Protozoa and algae

Samples of microplankton and larger nanoplankton (5 to 200 m) are preserved with a combination of Lugol's solution and DaFano's fixative. Organisms are then settled on a gelatin-coated slide, dried and embedded in 40 percent glycerin. Counting and sizing is performed under a microscope using a drawing tube, which facilitates measuring the organisms with a microcomputer-interfaced caliper. An interactive computer program, written in BASIC, allows for estimating the volumes of cells in up to 40 shape/species categories. The program then saves data on a disk, retrieves them, and calculates the results either for individual species (abundance, biomass, and mean cell volume) or as a pooled size spectrum of all organisms measured.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein.

The amino acid sequence of the 13-kDa polypeptide (P116) encoded by the first gene of the trfA operon of IncP plasmid RK2 shows significant similarity to several known single-stranded DNA-binding proteins. We found that unregulated expression of this gene from its natural promoter (trfAp) or induced expression from a strong heterologous promoter (trcp) was sufficient to complement the temperature-sensitive growth phenotype of an Escherichia coli ssb-1 mutant. The RK2 ssb gene is the first example of a plasmid single-stranded DNA-binding protein-encoding gene that is coregulated with replication functions, indicating a possible role in plasmid replication.     

Calcium- and magnesium-binding properties of oncomodulin. Direct binding studies and microcalorimetry

Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+. Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique. These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively. In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition. Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+. Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site. It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site. Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1. The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin. The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site.     

Developmentally regulated lectin in dark versus white axolotl embryos

The white mutant of the Mexican axolotl, A. mexicanum, involves an ectodermal defect which prevents melanophore colonization. Endogenous lectins have been suggested to function in neural crest-derived melanophore adhesion in other animals. To determine if differences in endogenous lectins exist in dark and white axolotls during melanophore colonization, white and dark ectoderm and carcass tissues have been assayed for lectin activity at premigratory, early migratory, and late migratory neural crest stages. Lectin content (specific for D-glucosamine, N-acetyl-D-glucosamine and D-mannose) increases significantly during early migration only in dark ectoderm and white carcass tissues, whereas white ectoderm and dark carcass lectin activities remain close to premigration levels. Neural crest cells in these embryos are associated with regions of high lectin activity suggesting that the differences in endogenous lectins may be involved in establishment of the dark/white phenotype.     

Application of pattern recognition and feature extraction techniques to volatile constituent metabolic profiles obtained by capillary gas chromatography

The applicability of threshold logic units, a form of nonparametric pattern recognition, to the processing of metabolic profile data obtained by high-efficiency glass capillary column gas chromatography has been investigated. The test data included profiles of the volatile constituents of urine from normal individuals and from individuals with diabetes mellitus. A feature extraction algorithm allowed for dimensionality reduction and indicated the constituents most important in the normal versus pathological distinction. With an optimum number of dimensions, a normal versus pathological prediction rate of 93.75% was achieved. Gas chromatography—mass spectrometry was utilized to identify important profile constituents.     

Thin-layer chromatography of β-aminopropionitrile

Thin-layer chromatography of β-aminopropionitrile (BAPN) in acetone and 1 m ammonium hydroxide (9:1) allowed separation of that compound from amino acids present in rat-liver perfusion fluid without prior solvent extraction. Direct densitometry of the spots obtained with ninhydrin yielded satisfactory quantitation of β-aminopropionitrile present. Utilization of [¹⁴C]nitrile-labeled β-aminopropionitrile and concurrent analysis of cyanoacetic acid allowed almost complete accountability of BAPN added to isolated rat liver.