Leishmania mexicana pifanoi: Analysis of the Antigenic Relationships Between Promastigotes and Amastigotes by Gel Diffusion,Immunoelectrophoresis, and Immunoprecipitation1

ABSTRACT. The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

STUDIES ON GUINEA PIG OOCYTES : I. Electron Microscopic Observations on the Development of Cytoplasmic Organelles in Oocytes of Primordial and Primary Follicles

Oocytes in primordial and primary follicles of young adult guinea pig ovaries fixed in osmium tetroxide and embedded in Epon 812, have been observed by electron microscopy. The gradual differentiation of a series of cytoplasmic organelles has been correlated with the growth in size of the oocyte and the development of the follicular wall. The most immature primordial oocyte is morphologically a simple cell consisting of a large slightly eccentric nucleus, a few large spherical mitochondria, a profusion of granular cytoplasmic vesicles, and free RNP particles. At the primary follicle stage, abundant endoplasmic reticulum, clusters of mitochondria proliferating around a rosette formation, multiple Golgi complexes, vesicular aggregates forming cortical granules, and a profusion of microvilli have been differentiated. Concentrations of cytoplasmic organelles at the periphery of the oocyte in the primary follicle suggest that it is equipped for the absorption, utilization, and intracellular transport of material delivered to its surface membrane. The juxtaposition of components of the ultrastructure during the development and growth of this large cell appears to follow a precise pattern and provides an unusual opportunity to study the interrelationships of differentiating organelles.     

STUDIES ON PROTEIN UPTAKE BY ISOLATED TUMOR CELLS : I. Electron Microscopic Evidence of Ferritin Uptake by Ehrlich Ascites Tumor Cells

Ferritin, added to the incubation medium of ascites tumor cells, was used as an electron microscopic marker to study the uptake of large protein molecules by morphologically intact cells. A definite uptake could be detected after 1 hour of incubation in Tyrode bicarbonate solution containing 0.04 to 13.3 mg ferritin/ml. Ferritin was found in a variety of membrane-surrounded structures, suggesting that pinocytesis and related membrane movements are occurring under physiological conditions and can account for the penetration of intact macromolecules into isolated tumor cells. Supplementation of the medium with serum albumin (33 mg/ml) increased the average amount of ferritin per cell and per pinocytotic structure. Ferritin was strongly adsorbed by fragments of lysed cells, which were readily taken up by intact cells. Besides its role as carrier, this debris appeared to stimulate membrane movements. Only rare examples were found to suggest the release of ferritin from the pinocytotic structures into the cytoplasm. Thus, the disintegration of such structures cannot be considered an obvious step towards a rapid metabolic utilization of protein by the cell. Particles of colloidal gold presented to the cell under the same conditions were not taken up to any significant extent, thus providing good evidence for a selective ingestion of particles of comparable sizes.     

Rb86 as tracer for potassium: I. Uptake of Rb and K by rice plant in nutrient solution

Summary The successful use of Rb⁸⁶ as a tracer for potassium in rice nutrition studies is related to the relative uptake of these two nutrient elements and the estimation of potassium by Rb⁸⁶ analysis. Results obtained under nutrient culture condition showed that the ratio of rubidium: potassium in the substrate was similar to that in the plant and the amount of potassium determined by conventional method was comparable to that of radiochemical analysis of Rb⁸⁶.     

Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.     

Laser-flash absorption spectroscopy study of the competition between ferredoxin and flavodoxin photoreduction by Photosystem I in Synechococcus sp. PCC 7002: Evidence for a strong preference for ferredoxin

The competition between ferredoxin and flavodoxin for electrons from Photosystem I was analyzed by flash absorption spectroscopy of the photoreduction processes that take place in the presence of both acceptor proteins in vitro. Steady state photoreduction assays indicate a strong inhibition of the apparent flavodoxin photoreduction activities of Photosystem I in the presence of ferredoxin. Flash-absorption experiments carried out at 626 nm, a wavelength where the reduction of ferredoxin shows no spectral contribution, show that the photoreduction of oxidized flavodoxin and flavodoxin semiquinone are inhibited by ferredoxin in a quantitatively similar way. The experimental data can be satisfactorily described by a reaction model that assumes that both redox states of flavodoxin do not compete with ferredoxin for binding on PS I and that the binding equilibrium between ferredoxin and PS I is not changed in their presence. In contrast, a model which assumes that ferredoxin and flavodoxin actually compete for binding to PS I gives poor results. Similarly, experimental data observed in the presence of both redox states of flavodoxin can also be quantitatively described under the assumption that the binding equilibrium between flavodoxin semiquinone and PS I is not disturbed by oxidized flavodoxin. Taken together, this analysis shows that PS I favors ferredoxin over flavodoxin and flavodoxin semiquinone over oxidized flavodoxin. This behavior is in accordance with the values of the dissociation constants for complexes between PS I and its acceptors. However, in case of ferredoxin the observed preference is stronger than expected from these values, indicating that ferredoxin is almost absolutely preferred by PS I over flavodoxin and is always reduced first.     

The inhibition of murine lymphocyte mitotic responses by human and mouse sera: I. Evidence for a role of antibody-independent activation of the alternative complement pathway

The responses of mouse lymphoid cell cultures to mitogens such as concanavalin A or antigen were inhibited by the addition of small amounts of fresh human serum. This inhibitory effect was reduced by specific decomplementation procedures such as heating at 50 °C to inactivate factor B or absorption with zymosan at 17 °C to deplete properdin from the serum. Human factor-B preparations reconstituted the inhibitory effect lost from human serum preparations heated at 50 °C. These findings are interpreted to indicate a fundamental role of activation of the alternative complement pathway (ACP) as an underlying mechanism of inhibition. Additional experiments designed to demonstrate a role of natural antibodies activating the classical complement pathway, while successful in these respects, also provided confirmatory evidence for an antibody-independent role of the ACP. Furthermore, data derived from experiments utilizing mouse sera tested on syngeneic mouse lymphoid target cells, were qualitatively similar to the results obtained employing human sera. The data suggest that the functional activities of mouse lymphocytes in vitro are inhibited by antibody-independent activation of the ACP, implying that this pathway may exercise a role in regulating lymphocyte function.     

Limitation of Photosynthesis by Carbon Metabolism : I. Evidence for Excess Electron Transport Capacity in Leaves Carrying Out Photosynthesis in Saturating Light and CO(2)

It has been investigated how far electron transport or carbon metabolism limit the maximal rates of photosynthesis achieved by spinach leaves in saturating light and CO₂. Leaf discs were illuminated with high light until a steady state rate of O₂ evolution was attained, and then subjected to a 30 second interruption in low light, to generate an increased demand for the products of electron transport. Upon returning to high light there is a temporary enhancement of photosynthesis which lasts 15 to 30 seconds, and can be up to 50% above the steady state rate of O₂ evolution. This temporary enhancement is only found when saturating light intensities are used for the steady state illumination, is increased when low light rather than darkness is used during the interruption, and is maximal following a 30 to 60 seconds interruption in low light. Decreasing the temperature over the 10 to 30°C range led to the transient enhancement becoming larger. The temporary enhancement is associated with an increased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, and increased levels of triose phosphate and ribulose 1,5-bisphosphate. Since electron transport can occur at higher rates than in steady state conditions, and generate a higher energy status, it is concluded that leaves have a surplus electron transport capacity in saturating light and CO₂. From the alterations of metabolites, it can be calculated that the enhanced O₂ evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis, by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

Studies on the Growth, Flowering, and Production of Female Sterile Flowers as Affected by Different Levels of Foliar Potassium in Solanum sisymbrifolium Lam.: I. EFFECT OF POTASSIUM CONTENT OF THE PLANT ON VEGETATIVE GROWTH AND FLOWERING

As compared to a high level of foliar potassium, a low but nota deficient level promotes shoot elongation and flowering inS.sisymbrifolium.This is accompanied by formation of flowers with rudimentaryovaries in which the megagametophyte in the ovules aborts atthe two- or the four-nucleate stage. Plants having a higherK content do not bear the female sterile flowers. Inflorescencesof the high-K plants with 20 per cent fewer buds have 23 percent higher dry weight than inflorescences of low-K plants.The ratio of organic nitrogen to dry weight is not affected,indicating that the inflorescences of the high-K plants arebetter supplied with metabolites in general.     

Photosynthetic responses of leaves to water stress, expressed by photoacoustics and related methods : I. Probing the photoacoustic method as an indicator for water stress in vivo

The effect of leaf desiccation on the photosynthetic activities in vivo was probed by the photoacoustic method. The aim of this research was: (a) To study the photoacoustic signal per se in varied conditions in order to develop this tool as a probe for stress conditions in vivo. (b) To obtain results pertaining to electron transport activities in vivo, and confirm conclusions based on work with isolated chloroplasts, which could otherwise be the result of nonspecific damage occurring during their isolation. Leaf discs from tobacco (Nicotiana tabacum L.) were routinely used, with other species tested also for comparison. Rapid leaf desiccation caused changes in the low frequency photoacoustic signal, attributed both to the mechanism of signal transduction, influenced by changes in the structural parameters of the leaf, and to the direct (nonstomatal) inhibition of gross photosynthesis. The dependence of the photothermal part of the signal on the frequency indicated the presence of two photothermal components, one of which persisted only at low modulation frequencies (below about 100 Hz) and which largely increased with the desiccation treatment. This component was ascribed to a thermal wave which reaches the leaf surface. The other nonvariable photothermal component was ascribed to a thermal wave propagating from the chloroplasts to the surface of the mesophyll cell. Only this component is considered in the ratio of the O₂ signal to the photothermal signal, which is used to estimate the quantum yield of photosynthesis. The specific dependence of the latter ratio on the frequency yielded a comparative quantum yield parameter from its extrapolation to zero frequency, and also indicated stress induced changes in the diffusion of O₂ through the mesophyll cell, reflected by changes in its characteristic slope. The (zero frequency extrapolated) quantum yield was markedly reduced with the progression of the water stress, indicating the inhibition of (gross) phototosynthetic electron transport in vivo. This result was expressed even more emphatically by the stronger inhibition of the photochemical energy storage, obtained by photoacoustic measurements at a high modulation frequency.