Woronin bodies fromPenicillium chrysogenum: Isolation and characterization by analytical subcellular fractionation

Differential centrifugation of whole homogenates ofPenicillium chrysogenum, disrupted by a modified Ballotini bead method, resulted in the enrichment of Woronin bodies between 800g (5 minutes) and 6000g (10 minutes). Isolated Woronin bodies are membrane-bounded, electron-opaque, approximately spherical organelles, 0.11 to 0.29 μm in diameter. Woronin bodies have a buoyant density (ϱ) of 1.21 g cm⁻³ and S_20,w values of 6300 to 37,600 in sucrose gradients. Analytical subcellular fractionation of whole homogenates in a zonal rotor showed that Woronin bodies did not cosediment with marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (cytochrome c oxidase), or endoplasmic reticulum (NADPH cytochrome c reductase).     

Ribosomal RNA cistrons in Euglena gracilis

Euglena gracilis chloroplasts contain about 12 fg DNA of average density 1.686 g cm^?3 and 1.7 pg RNA. The large (1.1 × 10⁶ mol. wt) and small (0.56 × 10⁶ mol. wt) ribosomal RNA components are coded for by separate cistrons, both of which band at a density of 1.696 g cm^?3 in a CsCl gradient. About 6% of the chloroplast DNA codes for rRNA indicating that there are 240 cistrons for rRNA in each chloroplast or about three to six cistrons per chloroplast genome. Similar studies with rRNA from cytoplasmic ribosomes indicate that the cistrons for cytoplasmic rRNA band at a density of 1.716 g cm^?3, denser than that of the main-band DNA, and that there are 1000 cistrons for cytoplasmic rRNA per cell. Fractionation of E. gracilis DNA on CsCl gradients and subsequent hybridization experiments, as well as melting curves of DNA-RNA hybrids, show that chloroplast rRNA does not anneal specifically with either the cistrons for cytoplasmic rRNA or any DNA in the dark-grown cell, in contrast to those results found in some higher plants.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

A differential heat-conduction microcalorimeter for heat-capacity measurements of fluids

A heat-conduction calorimeter has been developed for measuring small changes in heat capacity of milligram samples of membrane lipid dispersed in water as a function of temperature. The operation of the instrument is based on the principle that the thermal response of the sample to a short (10 s), electrically generated heat burst is a function of the diffusivity of the sample. Modeling studies of the instrument's performance have revealed that the output response after the heat burst is a function of only the heat capacity, ϱC_p. Calibration of the instrument experimentally confirmed this behavior. This feature obviated the need to measure the thermal conductivity in order to determine ϱC_p from the diffusivity equation, η = γ/ϱC_p. The calorimeter has the following characteristics: reproducibility of loading: ± 400 μJ/C° · cm³; baseline stability: ± 10 μJ/C° · cm³ per 36 h; resolution (± 1 S.D.): ± 50 μJ/C° · cm³; sample size 600 μl.     

Separation of Individual Stages of Trypanosoma cruzi Grown in Cell Culture by Continuous Free-Flow Electrophoresis

The separation of extracellular protozoan parasites from host cells based on a difference in surface charge has been described. However, with Trypanosoma cruzi no method exists for the isolation of pure parasite stages from heterogeneous mixtures. Studies on the electrophoresis of mixed stage populations confirm significant surface charge density differences exist among epimastigotes, trypomastigotes, and amastigotes. In ascending order of electronegativity, amastigotes have the lowest charge density, try-pomastigotes next, followed by epimastigotes. A technique has been developed for the separation of purified populations of parasites based on these charge differences using a continuous free-flow electrophoresis apparatus. The separated populations are morphologically intact and maintain their infectivity to mice. This separation method is applicable for preparative and analytical isolation of pure stages of T. cruzi for biochemical and immunological studies.     

Induction of ϱ− mutants in Saccharomyces cerevisiae by guanidine hydrochloride: I. Genetic analysis

Guanidine hydrochloride (GuHCl) induced in Saccharomyces cerevisiae cytoplasmic petite mutants (ϱ⁻) of the suppressive type. However, it was unable to induce the neutral type, even after prolonged incubation or increased drug concentration. No correlation was found between the degree of suppressiveness and the time of incubation of yeast cells with guanidine hydrochloride. The suppressiveness of ϱ⁻ induced was not altered by further treatment with GuHCl, whereas it was reduced upon treatment with ethidium bromide (EtBr). Some mitochondrial genetic information was lacking in the ϱ⁻ mutants induced by GuHCl, as demonstrated by the loss of the gene for erythromycin resistance and by reduced buoyant density of mitochondrial DNA of some ϱ⁻. There was no correlation between the degree of suppressiveness of the ϱ⁻ induced by GuHCl and the bouyant density of the mutant mitochondrial DNA.     

Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics

Okanla E. O., Stumpf J. L. &; Dusanic D. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics. International Journal for Parasitology12: 251–256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosoma cruzi in three weekly injections of 1 × 10⁸ trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund's complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestans. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestans included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and blood-stream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infestans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

The cAMP signal from Dictyostelium discoideum amoebae.

Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10^?9 to 3.5 × 10^?7M. Populations of 10⁶ cells could amplify an initial cAMP concentration of 2.5 × 10^?9M with a low probability, while an initial cAMP concentration of 5 × 10^?8M always induced a response. An initial cAMP concentration of 1.2 × 10^?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 10⁷ molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10^?7M or more. The amplification of a 10^?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 10⁴ cells/cm², both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10^?13–10^?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation.     

Isolation of Plastids from Sunflower Cotyledons during Germination

Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm⁻³. During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm⁻³. During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm⁻³. These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm⁻³ during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm⁻³ in very early stages of germination, and their density increased to 1.265 g cm⁻³, when glyoxysomal enzymes reached maximum development.     

Bovine toxoplasmosis: experimental infections.

Three calves dosed per os with 0.75 × 10⁶, 0.75 × 10⁶ and 1.5 × 10⁶Toxoplasma oocysts developed high titres of Toxoplasma antibodies as measured by the indirect fluorescent antibody test and the dye test. Toxoplasma gondii was reisolated from 2 of these animals. Two calves given 1 × 10³ tissue cysts and 2 × 10⁶ tachyzoites intramuscularly did not develop such high fitres, but T. gondii was reisolated from the calf injected with tachyzoites.Of 4 pregnant cows given 7 × 10⁴, 7 × 10⁴ and 1.6 × 10⁵ oocysts and 1.5 × 10² tissue cysts only 2 developed significant levels of Toxoplasma antibodies. There was no evidence of Toxoplasma infection in the calves born by these cows.It was concluded that cattle do not readily acquire persistent T. gondii infections, and this may be due, in part at least, to early elimination of Toxoplasma from their tissues.     

First results obtained with a sweeping pulsed radar reflectometer in the Globus-M tokamak

A weeping pulsed radar reflectometer designed for measuring the spatial electron density distribution in the Globus-M spherical tokamak with a minor plasma radius of a=24 cm, a major radius of R=36 cm, a toroidal field of B _T=0.5 T, a plasma current of I _p=200 kA, and an average density of n=(3–10)×10¹³ cm^?3 is described. The reflectometer operation is based on the reflection of microwaves with a carrier frequency f from a plasma layer with the critical density n=(0.0111f)², where n is the electron density in units of 10¹⁴ cm^?3 and f is the microwave frequency in GHz. By simultaneously probing the plasma at different frequencies, it is possible to recover the electron density profile. Microwave pulses with different frequencies are obtained by frequency sweeping. To increase the range of measured densities, channels with fixed frequencies are also used; as a result, the instrument has eleven frequency channels: a 19.5-GHz channel, eight channels in the 26-to 40-GHz frequency range, a 51.5-GHz channel, and a 60-GHz channel, which corresponds to eleven points in the density profile: 0.47×10¹³ cm^?3, eight points in the (0.8–1.95)×10¹³-cm^?3 range, 3.27×10¹³ cm^?3, and 4.5×10¹³ cm^?3. The reflectometer allows detailed measurements of the density profile with a time resolution of several tens of microseconds, which can be useful, in particular, in studying the processes related to the formation of an internal transport barrier in plasma. The first results obtained using this reflectometer in the Globus-M tokamak under various operating conditions are discussed.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Trypanosoma cruzi: cultivation in macromolecule-free semisynthetic and synthetic media.

A macromolecule-free semi-synthetic medium (F-81) was devised to culture Trypanosoma cruzi serially at room temperature. F-81 contains only one undefined substance, trypticase, which consists primarily of short-chain polypeptides. In F-81 medium T. cruzi will grow to a density of 35 to 43 × 10⁶ organisms/ml, a density comparable to that obtainable in a serum-containing medium such as F-69. High concentrations of water-soluble vitamins appear to have a serum-replacing effect in the F-81 medium. A completely synthetic medium (F-84) was prepared by replacing trypticase in F-81 with Trager's amino acid mixture. T. cruzi epimastigotes could be serially cultured in F-84, with a maximum yield of 9.2 × 10⁶ organisms/ml of medium after 3 to 4 weeks of incubation at 27 C.     

Nuclear buoyant density determination and the purification and characterization of wild-type neurospora nuclei using percoll density gradients

A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization

Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 × 10⁶ cells/cm² and upon confluency were reseeded as either low density (50 cells/cm²) or high density (8 × 10⁴ cells/cm²) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system.     

Potassium transport in normal and transformed mouse 3T3 cells

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in exponential and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 × 10⁴ to 4 × 10⁵ cell cm^?2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 × 10⁴ to 1 × 10⁶ cell cm^?2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.