Effect of a broad-host range plasmid on growth dynamics of Escherichia coli and Pseudomonas putida

Host-plasmid interactions were studied for the broad-host range plasmid, pTJS26, a derivative of RK2. To isolate host and plasmid contributions to the growth dynamics and plasmid stability, separate experiments were performed with host and recombinant cells for two different gram-negative hosts, Pseudomonas putida and Escherichia coli, at two different temperatures, 30 and 37 degrees C. At the lower temperature (30 degrees C) the growth kinetics were not affected by the plasmid, but plasmid instability was observed. At the higher temperature (37 degrees C) growth rates and yields were lower than that for the hosts, but the plasmid was stable. This behavior can be explained by a combination of two phenomena. First, the copy number control mechanism may be temperature sensitive and, second, plasmid segregation may be inefficient. For both E. coli and P. putida the growth dynamics of the recombinant system was dictated by the presence of the plasmid.     

Larval and adult morphology ofCamallanus anabantis Pearse, 1933 andC. kulasirii (Yeh, 1960) (Nematoda: Camallanidae) from freshwater fishes,with notes on the validity of some related forms

Larvae and adults ofCamallanus anabantis andC. kulasirii, recovered from the West Bengali freshwater fishes,Anabas testudineus andOphicephalus punctatus, respectively, are described on the basis of detailed morphological studies under the light microscope. Larval forms collected fromA. testudineus are deemed to be of the third and fourth stages when compared with those from experimental studies of the life cycle ofC. anabantis. Moreover, the present fourth stage female larvae are similar to the females ofC. pearsei, both morphologically and metrically.C. pearsei is, therefore, believed to represent the fourth stage female larvae ofC. anabantis. Similarly, adult males and females recovered fromO. punctatus closely resembleC. kulasirii andC. fernandoi, respectively. The larval forms from this host are fourth stage and can be distinguished as males and females, but both possess a buccal capsule bearing beaded longitudinal ridges similar to that of adult males. The late fourth stage (just prior to the final moult) female larva is, however, found to possess a buccal capsule transitional between that of the adult male and female and also betweenC. kulasirii andC. fernandoi. C. fernandoi is, therefore, presumed to represent the females ofC. kulasirii. However,C. pearsei andC. fernandoi are regarded, for the present, asspecies inquirendae.     

Mitochondrial genome in animal cells. Structure, organization, and evolution

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.     

d-Xylanase produced by Schizophyllum radiatum

Summary d-Xylanase (1,4--xylan xylanohydrolase, EC 3.2.1.8) was obtained from mycelial submerged culture of the mushroom Schizophyllum radiatum, grown on wheat straw pretreated with steam explosion as the substrate. The enzyme was purified 192-fold (specific activity 455 IU mg^-1 protein), with 37% yield with respect to total d-xylanase activity. Polyacrylamide electrophoresis of the d-xylanase peak showed a single band of protein whose molecular weight, calculated by electrophoretic mobility, was 25 700. The enzyme exhibited maximum activity at pH 4.9 and 55°C. d-Xylanase was stable from pH 5.0 to 7.5; its half-life was 12 h at 45°C. The Michaelis constant was 9.5 mg ml^-1 and V _max 0.37 mole min^-1. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylobiose, d-xylotriose, d-xylotetraose, and d-xylopentose showing the mode of action of an endo-type enzyme.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Growth Inhibition, Turgor Maintenance, and Changes in Yield Threshold after Cessation of Solute Import in Pea Epicotyls

The dependence of stem elongation on solute import was investigated in etiolated pea seedlings (Pisum sativum L. var Alaska) by excising the cotyledons. Stem elongation was inhibited by 60% within 5 hours of excision. Dry weight accumulation into the growing region stopped and osmotic pressure of the cell sap declined by 0.14 megapascal over 5 hours. Attempts to assay phloem transport via ethylenediaminetetraacetate-enhanced exudation from cut stems revealed no effect of cotyledon excision, indicating that the technique measured artifactual leakage from cells. Despite the drop in cell osmotic pressure, turgor pressure (measured directly via a pressure probe) did not decline. Turgor maintenance is postulated to occur via uptake of solutes from the free space, thereby maintaining the osmotic pressure difference across the cell membrane. Cell wall properties were measured by the pressure-block stress relaxation technique. Results indicate that growth inhibition after cotyledon excision was mediated primarily via an increase in the wall yield threshold.     

Microspectrofluorimetric analysis of the formaldehyde-induced fluorophores of 5-hydroxytryptamine and dopamine in intrapulmonary neuroepithelial bodies after administration ofl-hydroxytryptophan andl-DOPA

Summary To demonstrate the intracellular store of 5-hydroxytryptamine and dopamine in pulmonary neuroepithelial bodies of the neonatal rabbit after treatment with the corresponding amino-acid precursorsl-5-hydroxytryptophan orl-3,4-dihydroxyphenylalanine, formaldehyde-induced flourescence in combination with microspectrofluorimetric analysis has been used. Emission spectra and excitation spectra in an extended wavelength range from 240 to 460 nm, the displacement of excitation peaks after exposure to hydrochloric acid vapour, and calculation of peak ratio values 410/260, 380/320, 320/260 for phenylethylamine fluorophores and 385/315 for indolylethylamine fluorphores were performed. Thus, the presence of 5-hydroxytryptamine without occurrence of 5-hydroxytryptophan was demonstrated in pulmonary neuroepithelial bodies after administration of the corresponding biological precursor, while dopamine combined with 5-hydroxytryptamine were clearly revealed after administration ofl-3,4-dihydroxyphenylalanine. The rate of photodecomposition always corroborated these findings.Dedicated to Prefessor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Structure-from-motion based on information at surface boundaries

Existing computational models of structurefrom-motion — the appearance of three-dimensional motion generated by moving two-dimensional patterns — are all based on variations of optical flow or feature point correspondence within the interior of single objects. Three separate phenomena provide strong evidence that in human vision, structure-from-motion is significantly affected by surface boundary cues. In the first, a rotating cylinder is seen, though no variation in optical flow exists across the apparent cylinder. In the second, the shape of the bounding contour of a moving pattern dominates the actual differential motion within the pattern. In the third, the appearance of independently moving objects changes significantly when the boundary between them becomes indistinct. We describe a simple computational model sufficient to account for these effects. The model is based on qualitative constraints relating possible object motions to patterns of flow, together with an understanding of the patterns of flow that can be discriminated in practice.