Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes.

Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.     

Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16.

The four pectate lyases (EC 4.2.2.2) secreted by Erwinia chrysanthemi EC16 have been individually produced as recombinant enzymes in Escherichia coli. Oligogalacturonates formed from polygalacturonic acid during reactions catalyzed by each enzyme have been determined by high-performance liquid chromatography analysis. PLa catalyzes the formation of a series of oligomers ranging from dimer to dodecamer through a random endolytic depolarization mechanism. PLb and PLc are trimer- and tetramer-generating enzymes with an identical combination of endolytic and exolytic mechanisms. PLe catalyzes a nonrandom endolytic depolymerization with the formation of dimer as the predominant product. The pectate lyases secreted by E. chrysanthemi EC16 represent a battery of enzymes with three distinct approaches to the depolymerization of plant cell walls.     

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.     

Development and application of new positively charged filters for recovery of bacteriophages from water.

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.     

Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.     

Cell-substrate interactions during amoeboid locomotion of neutrophil leukocytes

Cell-substrate interactions between human blood neutrophils moving on a glass substrate in serum-free medium have been investigated using reflexion interference microscopy, high voltage and scanning electron microscopy (SEM). The contact pattern with the substrate differed considerably from that found in fibroblasts and the amoeba Naegleria. Discrete focal contacts could not be detected but large broad areas of very close contact (accounting for about 30% of the total contact area) could be found particularly associated with the uroid. Considerable loss of membrane material occurred as a result of breakdown of the uroid during locomotion.