Effects of Iron-, Manganese-, or Magnesium-Deficiency on the Growth and Morphology of Euglena gracilis1

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Histone formation, gene expression, and zinc deficiency in Euglena gracilis

Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.     

Accumulation of Extracellular Amino Acids by Euglena gracilis

SYNOPSIS. Normal Euglena gracilis , strain z, growing in the light in defined medium (initial nitrogen concentration 140 μ/ml) depletes the medium of all ninhydrin-positive N by the time a cell density of 2 million per ml is reached. A further 2- to 3-fold increase in the cell number takes place in the absence of exogenous N. The N content of an early log phase cell is about 100 picograms but decreases very rapidly as the culture continues to grow, reaching 22 picograms in the stationary phase. When grown in the dark, normal cells take up N somewhat more slowly but the supernatant fluid from saturation cultures is again devoid of N.
At modest cell densities, the permanently bleached strains examined contain less N per cell than do normal strains. The cultures of the bleached strains achieve a maximum density of about 1 to 2 million per ml rather than the 4 to 5 million reached by the normal strain. As a result, supernates from stationary phase cultures of bleached cells still contain a large proportion of the total N supplied.
Paper chromatographic analysis of these supernates reveals several ninhydrin-positive compounds. Most of these have been identified as common amino acids. Some of the properties of two unidentified, ninhydrin-positive compounds are described.     

Demonstration of enkephalin-, substance p- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

Summary The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of norphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Demonstration of enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of morphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

Regulation of catalase synthesis in Saccharomyces cerevisiae by carbon catabolite repression.

Summary A number of strains of Saccharomyces cerevisiae, wild type or respiratory deficient, were grown on glucose, galactose or raffinose. Specific activities of catalase T were about tenfold higher in late stationary wild type cells grown on glucose than in wild type cells harvested when glucose had just disappeared completely from the medium, or in respiratory deficient strains (rho^–, mit^–, pet) grown to stationary phase.Catalase A activity is completely absent in wild type cells grown to zero percent glucose or in respiratory deficient cells grown on glucose to stationary phase. High catalase A activity was detected in derepressed wild type cells and in a strain carrying the op 1 (pet 9) mutation, although this strain is unable to grow on nonfermentable carbon sources. All respiratory deficient strains tested have low, but significant catalase A activities after growth on galactose or raffinose.Wild type cells harvested during growth on glucose and rho^–-cells grown on low glucose to stationary phase contain enzymatically inactive catalase A protein. The apoprotein of the enzyme is apparently accumulated in rho^–-cells whereas glucose-repressed wild type cells seem to contain a mixture of apoprotein and heme-containing catalase A monomer.These results show that a source of chemical energy, probably ATP, is required for derepression of yeast catalase from catabolite repression. At least in the case of catalase A, energy produced by respiration is necessary if catabolite repression is caused by glucose. If less repressing sugars are utilized, ATP derived from fermentation appears sufficient for partial derepression. Formation of the active enzyme can apparently be influenced by carbon catabolite repression at different points: (1) at the level of protein synthesis, (2) at the stage of heme incorporation, (3) at the level of formation of the enzymatically active tetramer.     

Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography

Track autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient-enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track autoradiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.     

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle.

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates. Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growt rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analysis, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.     

THE ULTRASTRUCTURE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The freshwater dinoflagellate Glenodiniopsis steinii Wolsoszyńska was examined using computer-assisted three-dimensional reconstruction of serially sectioned cells observed with the transmission electron microscope and images from the scanning electron microscope. Vegetative cells contain ultrastructure typical of freshwater dinoflagellates including trichocysts, mitochondria, Golgi bodies, starch grains, and lipid bodies. The chloroplast is a single, multilobed structure, not multiple discoid chloroplasts as previously described. The “C” shape of the nucleus is apparently due in part to the size and location of the pusule.     

Effects of cell surface damage on surface properties and adhesion of Pseudomonas aeruginosa

Bacterial cell surfaces play a crucial role in their adhesion to surfaces. In the present study, physico-chemical cell surface properties of Pseudomonas aeruginosa, isolated from a case of contact lens associated keratitis, are determined for mid-exponential and early stationary phase cells and for cells after exposure to a lens care solution or after mechanical damage by sonication. Exposure to a lens care solution and mechanical cell surface damage reduced the cell surface hydrophobicity and water contact angles decreased from 129 degrees to 96 degrees and 83 degrees, respectively. Zeta potentials in saline (-9 mV) were hardly affected after mechanical damage, but tri-modal zeta potential distributions, with subpopulation zeta potentials at -11, -28 and -41 mV, were observed after exposure of bacteria to a lens care solution. X-ray photoelectron spectroscopy indicated changes in the amounts of oxygen-, nitrogen- and phosphorus-rich cell surface components. Mid-exponential phase cells had more nitrogen-rich cell surface components than early stationary phase cells, but water contact angles and zeta potentials were not very different. In addition, mid-exponential phase cells adhered better than early stationary phase cells to hydrophobic and hydrophilic substrata in a parallel plate flow chamber. The capacity of P. aeruginosa to adhere was decreased after inflicting cell surface damage. Exposure to a lens care solution yielded a larger reduction in adhesion capacity than sonication, likely because sonication left most of the cells in a viable state, in contrast to exposure to a lens care solution. It is argued that for clinically relevant experiments, it may be preferable to work with surface damaged cells rather than with gently harvested organisms.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

ULTRASTRUCTURE OF THE CARPOSPOROPHYTE AND CARPOSPOROGENESIS IN THE PARASITIC RED ALGA PLOCAMIOCOLAX PULVINATA SETCH, (GIGARTINALES,PLOCAMIACEAE) 1

The ultrastructure of the carposporophyte and carposporogenesis is described for the parasitic red alga Plocamiocolax pulvinata Setch. After presumed fertilization the zygote nucleus is apparently transferred to the auxiliary cell which initiates gonimoblast cell production. These gonimoblast cells differentiate into storage or generative cells. Storage gonimoblast cells (SGC) are large and multinucleate, contain large quantities of starch and are located nearest the auxiliary cell, when compared to the smaller uninucleate, devoid of starch, generative gonimoblast cells (GGC) that form terminal lobes of carpospores. In addition, compressed membrane bodies and annulate lamellae are common in these cells. During carposporophyte maturation the amount of starch in the SGC's decreases and eventually the auxiliary cell, as well as SGC's, degenerate. Generative gonimoblast cells (GGC's) cleave repeatedly to form carpospores which are interconnected by small pit connections. Stage one-carpospores are recognized by their elongated shape, the formation of small     

Ultrastructural, physiological, and cytochemical characterization of cores in group D streptococci.

Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 mum in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Glumatic-Oxaloacetic Transaminase in Leaves of Phosphorus-Deficient Citrus Plants

Phosphorus deficiency in citrus leaves resulted in reduced glutamic-oxaloacetic transaminase (GOT) activity and low pyridoxal-phosphate (PLP) content. GOT activity was estimated in crude enzyme extracts by spectrophotometry PLP content was detected colorimetrically in water-alcoholic extracts. K and Cu deficiencies increased; -N, -S and -Zn decreased and -Mg, -Fe and -Mn did not affect GOT activity in citrus leaves. Experiments were conducted to restore enzyme activity either by direct addition of PLP to the reaction mixture or by infiltration of PLP or KH₂PO₄ to detached, intact leaves. The infiltration was carried out in vacuo and the leaves were incubated on wet paper for 26 h, after which enzyme activity was estimated. Transamination activity of -P leaves more restored by PLP than by KH₂PO₄ treatments. In zinc-deficient leaves the enzyme activity was not restored by infiltration of KH₂PO₄.