Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Str?m, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

Assimilation of animal fats by yeasts and synthesis of protein by continuous cultivation in a complex nutrient medium

Under the conditions of continuous cultivation studies were carried out on a mixed culture (association) with two strains of yeasts: Candida sp., assimilating animal fats, and Candida scottii, assimilating reducing sugars. A complex nutrient medium of wood hydrolysate was used. It was established that in this medium, at a rate of dilution of D = 0.25 h^?1, the mixed culture assimilates the fats and the sugars simultaneously. As a result of this the yield of absolutely dry yeasts with regard to the total sum of fats and sugars amounts to 71% in the case of incineration fat and to 82% in the case of technical pork fat. The contents of protein, amino acids, lipids and ashy substances in the yeasts obtained from a medium with fats do not differ substantially from those obtained from the same medium without fats.     

Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy

We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.     

The Stroop effect on Slovak-Hungarian bilinguists

We studied the processes of interference vs language automatisms in a group of Slovak-Hungarian bilinguists (N = 19, age 13 years) and compared their performance in the Stroop test with two control groups - Slovak (N = 24) and Hungarian (N = 31) monolinguists of the same age. - The Stroop effect, whose essence is an interfering effect of the meaning of the word on its physical characteristic - colour, proved to be a suitable method in the study of information processing in the semantic memory of bilinguists as well as in determining dominance of one language over the other. Slovak-Hungarian bilinguists showed, as a rule, shorter times in Hungarian and approximated in their performance Hungarian monolinguists. It can be assumed that reaction times in the Stroop test are conditioned not only by interference of the other language but also by language automatisms, whereby interference and speech automatisms are in an indirect correlation. - The results thus indicate that processes of interference and language automatisms cannot be separated. Dominance of one language structure over the other (Hungarian over Slovak) was confirmed not only in our experimental method but also in the subjective rating.     

Reconstitution of in vivo cell-mediated lympholysis responses in aged mice with interleukin 2

Advancing age is accompanied by declining immune potential. Both humoral and cellular immune responses are diminished in aged humans and experimental animals. A major lesion preventing effective T cell-mediated responses is the lack of interleukin 2 (IL 2) synthesis in aged mice. Addition of IL 2 can effectively reconstitute in vitro T cell-dependent humoral and cell-mediated immunity. These results have been extended, demonstrating that IL 2-containing lymphokine preparations when administered with antigen can restore the ability of aged mice to generate cytotoxic T lymphocytes in vivo. Furthermore, IL 2 has been identified as the active component of these lymphokine preparations through the use of purified human IL 2 prepared by recombinant DNA and gene cloning technology. The cloned IL 2 is highly effective in enhancing the immune responses of aged animals both in vitro and in vivo.     

gamma-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters

Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.     

Biochemical changes in the rat during experimentally induced acute ochratoxicosis

Biochemical changes in rat urine and tissues treated with five consecutive daily doses of ochratoxin A (10 mg/kg body weight) were studied. Urine volume and urinary proteins were moderately raised during the first few days of ochratoxin treatment, and were then highly elevated towards the end of the investigation. Urinary muramidase excretion was significantly raised (p less than 0.01) 24 h after the first insult with the toxin. The urinary output of alkaline and acid phosphatases, lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) were all elevated but very much later, during the course of injections with ochratoxin A. Kidney alkaline and acid phosphatases, LDH and GDH were correspondingly reduced 7 days from the beginning of ochratoxin A administration. Liver LDH activity was reduced while serum LDH was raised. Liver glycogen level was significantly (p less than 0.0001) increased. Experimental evidence was presented to show that the initial point of interaction of ochratoxin A with the rat renal system may be at the first portion of the proximal convoluted tubular cell region.     

Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.     

Mutability of the LYS2 gene in diploid saccharomycete yeasts. I. The occurrence of spontaneous mutants and mutants induced by x-ray and ultraviolet radiation

More than 3000 spontaneous and induced lys2 mutants were obtained in haploid and diploid strains of yeast Saccharomyces. The ability to utilize alpha-aminoadipate was used for lys2 mutant screening. The spontaneous and induced mutation rates were measured in haploid and diploid strains. Mitotic segregation of pho1 marker linked to LYS2 was studied in lys2 mutants obtained in diploid strains. Fertility of diploid lys2 mutants was tested. The conclusion to be drawn from the data presented is that mutations appeared in one of two homologous chromosomes and then segregated by mitotic homozygotization.