Cyclic AMP levels in migrating and non-migrating newt epidermal cells

Cyclic AMP (cAMP) levels were measured in 8-hour migrating wound epithelial and non-migrating epithelial cells of the newt. Tissues were collected in vivo and in vitro with and without epidermal-dermal separation by collagenase. Regardless of manner of collection and treatment, cAMP levels were always significantly higher in the migrating cells. Levels were also measured in 28-hour and 36-hour wound epithelia. There was a progressive decline in levels in wound epithelia between 8, 28, and 36 hours, suggesting that levels were in the process of returning to normal. When cells were treated with a dose of cAMP and theo-phylline previously shown to inhibit migration, levels of cAMP were much higher than any migrating epithelium. The fact that cAMP inhibits migration, yet migrating cells have higher cAMP levels, seems contradictory at first, but possible explanations are advanced to account for the apparent discrepancy.     

Use of fluorescently labelled algae to measure the clearance rate of the rotifer Keratella cochlearis

1. Fluorescently labelled algae (FLA) were used to measure clearance rates of the rotifer Keratella cochlearis. The freshwater algae Chlorella vulgaris and Stichococcus bacillaris were labelled with a fluorescent dye, 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), following a modified staining procedure. 2. Keratella cochlearis ingested both algal species. Clearance rates on tracer foods varied between 2.4 and 6.9 μl ind^?1 h^?1, which are comparable with those determined using other techniques. 3. The main drawback of the FLA technique was that only a little more than one-third of the total amount of algal cells of both C, vulgaris and S. bacillaris were well stained with the dye (DTAF), despite the use of a higher concentration of dye and a longer staining period than recommended in the literature. 4. The FLA method can be successfully applied in grazing studies involving size selection and competition for food among zooplankton. The method complements existing techniques for measuring the clearance and ingestion rates of filter-feeders.     

Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells

Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin-binding motor protein, as a major G-actin-interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile-based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.     

Growth of migrating and non-migrating cryptophytes in thermally and chemically stratified experimental columns

• 1 Growth rates of migrating and non-migrating populations of two strains of freshwater cryptophytes, CCAP 979/67 and 979/62, under different light and nutrient regimes were calculated from experiments conducted in laboratory columns which were thermally stratified. During the experiments, cellular carbon, nitrogen, phosphorus, carbohydrate and protein were also analysed. The intention was that the populations would become either phosphorus- or nitrogen-depleted following a period of growth.
• 2 In all experiments, populations of cryptophytes grew but growth appeared of short duration. In a phosphorus depletion experiment with Cryptomonas 979/67, there was a period of rapid growth starting on day 2 and finishing on day 8, during which the estimated growth rate was c. 0.9 div. day^-1. In a nitrogen depletion experiment, the period of rapid growth of C. 979/67 lasted only for 2–3 days with a growth rate of c. 0.85 div. day^-1.
• 3 In a phosphorus depletion experiment with C. 979/62, the onset of a period of rapid growth coincided with the commencement of diel vertical migration. The highest growth rate was estimated as c. 1.0 div. day^-1. In a nitrogen depletion experiment, C. 979/62 did not migrate and attained a growth rate of only 0.28 div. day^-1.
• 4 For C. 979/67 the highest observed growth rate was lower than the maximum potential growth rate of 1.38 div. day^-1 estimated in batch culture. For C. 979/62 the maximum growth rate in the column was similar to the maximum potential growth rate of 0.87 div. day^-1 in batch culture experiments.
• 5 The results suggest that some migrating cryptophytes under favourable conditions in stratified water columns can attain high growth rates supporting the hypothesis of Raven & Richardson (1984) that, based on cost-benefit analysis, diel vertical migrations could increase the growth rate of flagellates. Such growth appears of short duration and its ecological importance still requires further verification.

     

Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria.

Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA. The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell. Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls. A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only. Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei. Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L. lactis and M. phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.     

Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex.

Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.     

Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin beta 4 in migrating IEC-6 cells

The cause of reduced migration ability in polyamine-deficientcells is not known, but their actin cytoskeleton is clearly abnormal.We depleted polyamines with -difluoromethylornithine (DFMO) inmigrating cells with or without stimulation by epidermal growth factor(EGF) and investigated filamentous (F-) actin, monomeric (G-) actin,and thymosin 4 (T4), using immunofluorescent confocal microscopy,DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cellinterior, increased it in the cell cortex, redistributed G-actin, andincreased nuclear staining of T4. However, DFMO did not affect theamount of T4 mRNA. EGF caused a rapid increase in the staining ofF-actin in control cells, but DFMO prevented this response to EGF.Despite the visible changes shown by immunocytochemistry, statisticallysignificant changes in the amount of either actin isoform or of totalactin did not occur. We propose that DFMO reduces migration byinterfering with the sequestration of G-actin by T4 and theassociation of F-actin with activated EGF receptors.     

Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells

BACKGROUND: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.     

Binding of deoxyribonuclease I to actin: a new way to visualize microfilament bundles in nonmuscle cells.

Intracellular actin-containing fibers can be visualized by indirect immunofluorescence microscopy when they are stained with antibody directed against DNase I. The location of actin-containing fibers in cells appears to be similar to the staining pattern of antibody to actin. Actin fibers were also visualized by direct fluorescent microscopy with rhodamine-conjugated DNase I.     

A new method of synthesis of fluorescently labelled oligonucleotides and their application in DNA sequencing.

A new approach to the chemical synthesis of oligodeoxynucleotides bearing reporter functional groups at base residues of 3'-end nucleosides is reported. Applications of the 3'-end fluorescently labelled primers for automated DNA sequencing are shown.     

Cell-free synthesis of the Ras-binding domain of c-Raf-1: binding studies to fluorescently labelled H-ras.

It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.     

Swimming capacity and energetics of migrating and non-migrating morphs of three-spined stickleback Gasterosteus aculeatus L. and their ecological implications

The two morphotypes (leiurus and trachurus) of the three-spined stickleback Gasterosteus aculeatus , caught at the same location and time in the River Scheldt (Belgium), were investigated for physiological differences in swimming capacity and energetics associated with migration. Critical and optimal swimming speeds, maximum speed and gait-transition speed were significantly higher for the trachurus type. Standard metabolic rate and active metabolic rate were also higher for trachurus, as was scope for activity. Energy stores (protein, lipid and glycogen in liver and white muscle) were mostly similar in the two types, but lipids in trachurus liver tissue were significantly higher.     

Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides.

rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals (clay minerals) and organic material (up to 8%) did not significantly interfere with signal expression after hybridization. Background signals were mainly caused by autofluorescence of organic material. Non-specific binding of labelled oligonucleotides to soil particles was not observed. In situ detection of introduced cells of Pseudomonas cepacia in a sandy loam spiked with a mixture of selected soil micro-organisms was possible after hybridization with a specific probe. Analysis of natural bacterial populations in soil, however, was not possible by in situ hybridization without activation of these micro-organisms by adding nutrients. Growing cells, e.g. Streptomyces scabies hyphae growing in amended soil, were easily detected.     

Alkaline deoxyribonuclease activity in mouse teratocarcinoma cells: variation of enzyme levels during differentiation.

Embryonal carcinoma (EC) cells contain an alkaline DNase whose specific activity is much higher than their differentiated derivatives. After partial purification on CM-Sephadex, fractions eluted at 0.15 M NaCl contain a DNase activity which is inhibited by G-actin. The possible role of this alkaline DNase activity in maintaining the unpolymerized state of actin filaments in EC cells is discussed.     

Purification and properties of human pancreatic deoxyribonuclease I.

Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.     

Inhibition of deoxyribonuclease I by actin is to protect cells from premature cell death

Deoxyribonuclease I (Dnase1) is the major extracellular endonuclease. It is secreted by digestive glands into the alimentary tract and into the plasma, lacrimal fluid and urine by hepatocytes, lacrimal glands and renal proximal tubular cells, respectively. In many species the activity of Dnase1 is inhibited by monomeric actin. However, the biological significance of this high affinity interaction is unknown. We generated a Dnase1 mutant with extremely reduced actin binding capacity. EGFP-constructs of wild-type and mutant Dnase1 were transfected into MCF-7 breast cancer cells and apoptosis or necrosis was induced by staurosporine or oxidative stress. During apoptosis faster chromatin fragmentation occurred in cells transfected with mutant Dnase1. When wt (wild-type)- or mutated Dnase1 were added to cells after induction of necrosis, faster chromatin degradation occurred in the presence of mutant Dnase1. Inclusion of actin under these conditions inhibited chromatin degradation by wt- but not by mutated Dnase1. Thus, inhibition of Dnase1 by actin may serve as a self-protection mechanism against premature DNA degradation during cell damage.     

Crystallization of cytoplasmic actin in complex with deoxyribonuclease I.

Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.     

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.

BACKGROUND: Two calibration methods have been proposed for determining the relation between the fluorescence ratio of a pH-sensitive fluorescent indicator and intracellular pH (pHi). The first method uses nigericin to clamp pHi to external pH (pHe) and the second is the null point method. We compared these different calibration methods, solution conditions, and temperatures by using flow cytometry and the fluorescent dye 1,5- (and-6)-carboxy seminaphtorhodafluor-1-acetoxymethyl ester with an NS0 cell line. METHODS: The nigericin method was performed in glucose solutions supplemented with KCl and 2-(N-morpholino)ethane sulphonic acid plus tris(hydroxymethyl)aminomethane (solution 1A), a mixture of K2HPO4/KH2PO4 in glucose-solution supplemented solutions (solution 2A), or bicarbonate buffered growth medium supplemented with K2HPO4/KH2PO4 (solution 2B); this allowed a range of pHe values to be used. The effect of temperature (22 degrees C or 37 degrees C) on the nigericin calibration curve was also investigated. The null point method was performed by using a series of solutions with a mixture of weak acid and base with a known pHi response. RESULTS: Using solution 1A as the calibration solution resulted in acidic values of pHi for cells cultured in medium as compared with the values achieved with solution 2A. Using solution 2B did not affect the calibration curve. For the temperatures considered in this study, there was no affect on the calibration curve, but temperature did affect the pHi value of cells in phosphate buffered saline. The pseudo-null point method used with flow cytometry resulted in a calibration curve that was significantly different (P<0.05) from that achieved using the nigericin method. CONCLUSIONS: Our data indicates that the choice of calibration solution can affect the reported pHi value; therefore, careful choice of solution is important.