Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Isolation and properties of crystalline ferredoxin from lactuca sativa

Lettuce ferredoxin has been purified to homogeneity, with a yield of 18 mg/kg of denerved leaves. It crystallizes in magnificent needles, often clustered in broom-like sheaves. The absorption spectrum showed maxima at 460, 422, 330 and 274 nm,with a ratio A₄₂₂/A₂₇₄, of 0.46. The mM absorption coefficient was 9.74 at 422 nm, and 21.62 at 274 nm. This ferredoxin showed a pI = 4.7 and an E′₀ = ?425 mV (at pH = 7.7). MWs of 12 400, 11480 and 13000 were obtained by sucrose gradient centrifugation, and on the basis of the amino acid composition and the iron content, respectively, with an average of 12 300. The amino acid analysis showed the existence of one methionine residue per mole, with 105 amino acid residues. There are two iron atoms and two labile sulfide groups per mole; 4 half-cystine residues were found by performic acid oxidation, and 5 cysteine groups when determined by titration with pHMB. The native protein is not fixed on thiol-Sepharose 4B, but it is quantitatively retained after incubation with 8 M urea. Lettuce ferredoxin showed a 62, 58 and 78% effectiveness with the spinach ferredoxin-NADP reductase, nitrite reductase and fructose-1,6-diphosphatase (FDPase), respectively, when compared with the spinach ferredoxin. This different behaviour of both ferredoxins is joined to genetic-structural relationships, and suggests that the role of ferredoxin in FDPase activation is more sophisticated than that of a mere nonspecific reductant.     

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( _w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. _wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, _w was similar to the water potential of the environment when cell enlargement was not occurring. The low _w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (Lp). Despite the low Lp of growing cells, Lp decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and Lp increased when auxin was resupplied exogenously. The auxin-induced increase in Lp was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in Lp appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to Lp remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.     

Rhesus monkey micro mixed lymphocyte and mitogen reactivity: Optimal conditions and normal variability

Optimal conditions for the rhesus monkey micro mixed lymphocyte system with multiple automated harvesting of samples were evaluated. Parameters studied were cell concentration, length of culture period, methods of inactivation of cell populations, supplementation of media, type of culture plates, and changes in the reactivity of cells from individual animals over an extended time period. This work was supported in part by Portland Veterans Administration Hospital, Portland, Oregon, and the General Research Support Branch of the U.S. Public Health Service Grant RR00163, the Bureau of Medicine and Surgery, Navy Department, Work Unit No. M4318. 01.007ABG2. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the Naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89–54 as amended by Public Law 91–579, “Animal Welfare Act of 1970,” and the principles outlined in the “Guide for the Care of Laboratory Animals,” U.S. Department of Health, Education, and Welfare Publication No. (NIH) 73-23.     

Dence for a gene in rats affecting lymphocyte responsiveness to PHA

A gene controlling high responsiveness of lymphocytes to in vitro stimulation by PHA was transferred from the Lewis strain of rats to the BN background by ten generations of backcrossing. The high-responder phenotype was initially defined on the basis of incorporation of³H-thymidine, but we show that this trait also involves higher levels of mitotic activity than are observed with low responder lymphocytes. This gene is not closely linked to any histocompatibility locus which could be detected by skin grafting, and it does not appear to affect the proportion of T lymphocytes.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Cell growth and water relations of the halophyte,Atriplex nummularia L., in response to NaCl

Summary Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic over adjustment as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118–125).