Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

Enzyme assay in cultures of Escherichia coli by a continuous flow method based on lysis from without by a phage ghost.

Lysis of Escherichia coli from without by excess of phage ghost has been shown to give excellent yield of several enzymes. The application of lysis from without to a continuous determination of enzymes in growing cultures of E. coli is illustrated with β-galactosidase. This application can be used in studies on changes of a large number of enzymes during metabolic perturbation (induction, repression, etc.) of growing cultures of E. coli.     

Alterations in the characteristics of 2-deoxy D-glucose transport into cultured human glioma cells during cell growth.

The uptake of 2-deoxy-d-glucose (2-DG) into human glioma cells (138 MG) was related to cell growth. The uptake of 2-DG was high at confluency but low in both rapidly growing sparse cultures and in growth-inhibited dense cultures. Lineweaver-Burke plots of uptake at different cell densities showed changes in V_max; K_m, however, remained constant. Dibutyryl cyclic-AMP (db-cAMP) doubled the uptake of 2-DG into rapidly growing sparse cultures but lacked effect at higher cell density. Independent of their density, cells treated with db-cAMP attained the characteristic morphology of differentiated glial cells.     

Activity of thidiazuron inin vitro shoot cultures ofPrunus sp. andMorus alba

Thidiazuron incorporated into MS medium stimulated rosettes formation only in some treatments. This effect was more pronounced in cultures ofMorus alba thanPrunus sp. Mulberry cultures responded to the optimal concentration of thidiazuron (0.2 mg I^?1) not only with shoot formation but also, with growth of large leaves and poor development of callus tissue. In cultures of both investigated genera the shoot elongation was inhibited. Shoots of mulberry cultures growing on proliferation medium supplemented with thidiazuron formed roots, in many cases.     

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser³¹⁵ which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser³¹⁵ appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser³¹⁵ mutations are favoured in the slow growing cultures.     

Phosphorylase isoenzymes in Polytoma uvella

Starch phosphorylase isoenzymes in growing cultures of Polytoma uvella were isolated by gel electrophoresis and DEAE-cellulose chromatography. Two forms (A and B) were found. These were not detected in the lag phase of the cultures but both forms were present in the log and early stationary phase. Initially form A was more prominent than form B. In the early stationary phase, form A decreased and only form B could be found in the older cultures.     

Role of the ptsN Gene Product in Catabolite Repression of the Pseudomonas putida TOL Toluene Degradation Pathway in Chemostat Cultures▿

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIA^Ntr participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.     

Model system studies of the use of 5-bromo-2′-deoxyuridine for selection of deficient mutants in plant cell suspension and protoplast cultures

Experiments using separate growing and arrested cell suspensions showed that treatment with the appropriate level of BUdR selectively killed the growing cultures. This effect was not light-dependent. A model system comprising leaf protoplasts of 2 different mutants of Nicotiana tabacum, only one of which was sensitive to valine, was developed to test the ability of this method to select non-dividing from dividing cells in mixed cultures. Following treatment of cultures with BUdR in the presence of valine, colonies recovered were tested on media which clearly differentiated the cell types on the basis of valine resistance and the ability to green in the presence of streptomycin (the marker for the valine-sensitive line). BUdR treatment of valine-inhibited cultures increased the percentage of valine-sensitive colonies recovered from mixtures containing up to 99.9% valine-resistant cells, although recovery of sensitive colonies was poor where these cells only represented a small proportion of the culture.     

Factors Influencing Emergence of Juveniles from Cysts of Heterodera zeae

Several factors were studied to determine their effects on hatch and emergence of second-stage juveniles (J2) from cysts of Heterodera zeae. The optimum temperature for emergence of J2 from cysts of H. zeae was 30 C. No juveniles emerged from cysts at 10 or 40 C. Immersion of cysts in 4 mM zinc chloride solution stimulated 10% greater emergence of J2 than occurred in tap water controls during 28 days. Fresh corn rhizosphere leachates from 25-day and older plants growing in sand or sandy field soil stimulated 22-24% greater emergence of J2 from cysts than occurred in tap water after 28 days. Rhizosphere leachates stored for 30 days at 4 C and leachates of sand, sandy field soil, and silty field soil inhibited emergence of J2 from cysts by 7-12% compared to tap water. Rhizosphere leachates from corn plants aged 20, 30, 40, 50, or 60 days growing in sandy field soil stimulated emergence of J2 from cysts. Similar numbers of J2 emerged from cysts regardless of whether the source of cysts was field microplot cultures, greenhouse cultures, or growth chamber cultures. Fertilizing growth chamber cultures of H. zeae on corn plants resulted in a doubling of the numbers of cysts produced in the cultures, and those cysts yielded 2-3 times as many emerged J2 in hatching tests compared to cysts from similar unfertilized cultures.     

Induction of respiratory incompetent mutants by unsaturated fatty acid depletion in Saccharomyces cerevisiae

Constant levels of cellular unsaturated fatty acids were obtained by growing a fatty acid desaturase mutant of $\text{Saccharomyces cerevisiae}$ in glucose limited chemostat cultures supplemented with various concentrations of Tween 80. An increase in the frequency of cytoplasmic respiratory incompetent mutants was observed in cultures growing at low cellular levels of unsaturated fatty acids. This effect has been shown to result from an increase in the rate of mutation as the cellular unsaturated fatty acid level is decreased. The majority of induced petite mutants are ?° (contain no mitochondrial DNA).     

CK2 activity is modulated by growth rate in Saccharomyces cerevisiae

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k_cat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.     

Tapé fermentation

Microorganisms isolated from ragi, originally obtained from Indonesia, were selected for their ability to convert steamed glutinous rice into tapé, an Indonesian fermented food. A mixture of Chlamydomucor oryzae and Endomycopsis fibuliger had good fermentation characteristics. Prepared starters, produced by growing pure cultures on rice and drying them, were as active as pure cultures grown for 4 days on Difco mycological agar slants at 30 C.     

Mitochondriogenesis in synchronous cultures of yeast. I. Oscillatory pattern of respiration

Synchronised cultures of yeast, Saccharomyces cerevisiae provide ideal material for the study of mitochondria. While studying the oxygen uptake capacity of synchronously growing cultures, it was noticed that oxygen uptake increased and decreased in an oscillatory pattern. Such a pattern was repeated up to four cycles. This observed behavior is entirely different from the results reported earlier by others.     

Partial purification and properties of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase from higher plants

The partial purification and characterization of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) from two plant sources growing as cell cultures are reported. The enzymes have been purified 50-and 16-fold, respectively, and show an absolute specificity for p-coumaroyl-CoA and caffeoyl-CoA as well as for quinate, and are responsible for the synthesis of p-coumaroylquinate and caffeoylquinate (chlorogenic acid). The distribution of this transferase activity in a variety of plant cell cultures and differentiated plants is reported.     

Tapé Fermentation

Microorganisms isolated from ragi, originally obtained from Indonesia, were selected for their ability to convert steamed glutinous rice into tapé, an Indonesian fermented food. A mixture of Chlamydomucor oryzae and Endomycopsis fibuliger had good fermentation characteristics. Prepared starters, produced by growing pure cultures on rice and drying them, were as active as pure cultures grown for 4 days on Difco mycological agar slants at 30 C.     

Biology of Azospirillum-Sugarcane Association: Enhancement of Nitrogenase Activity

Azospirillum brasilense was reisolated from associations with callus tissue cultures of sugarcane and compared with stock cultures of the inoculated bacterium and related strains. Although the reisolate had a growth rate similar to stock cultures, it exhibited a severalfold increase in maximum specific activity of nitrogenase. The reisolate and the parent culture had similar ultrastructure. The general ultrastructure of Azospirillum is described. The bacterium was capsulated when grown on nitrogen-free nutrient agar plates and on callus, but was not capsulated when growing in a subsurface zone in N-free semisolid nutrient agar, except rarely in aging cultures. Capsulation may be a protective mechanism against unfavorable pO₂ under dinitrogen-fixing conditions. Pleomorphism occurred in capsulated forms, and the ultrastructure of these forms is described.     

Monoterpene metabolism in cultures of Rosa species

Callus and suspension cultures of Rosa damascena maintained under ranges of conditions that were predicted on the basis of current hypotheses to favour secondary metabolism accumulated negligible amounts of monoterpenes. However, enzymes that converted mevalonate and isopentenyl pyrophosphate into geraniol and nerol could be extracted from the apparently inactive callus with activities up to 100-fold greater than those from the parent plant, and these activities were optimum in cultures that were slow growing or were in the stationary phase. Callus and suspension cultures both rapidly metabolized exogenously supplied monoterpenes via oxidative pathways. Thus nonaccumulation may be due to degradation of nascent products formed endogenously. Similar results were obtained from less detailed studies on R. gallica, R. canina and R. cv. Paul's Scarlet.     

Animal cell cultures in microsporidial research: their general roles and their specific use for fish microsporidia

The use of animal cell cultures as tools for studying the microsporidia of insects and mammals is briefly reviewed, along with an in depth review of the literature on using fish cell cultures to study the microsporidia of fish. Fish cell cultures have been used less often but have had some success. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable in understanding and treating microsporidia infections in fish, which are increasingly of economic importance.