Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Synthesis and Release of Cyclic Adenosine 3':5'-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [³²P]cAMP was purified from cultures supplied [³²P]phosphate. The compound was identified as [³²P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a ³²P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a ³²P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [³H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Emission of ethylene and ethane by leaf tissue exposed to injurious concentrations of sulfur dioxide or bisulfite ion

Leaf tissues injured with SO₂ gas or bisulfite ion in solution emit ethylene and ethane. The amounts of these gases produced by the tissues depend on the degree of exposure to SO₂ or bisulfite. The amount of ethylene produced in response to SO₂ fumigation correlates positively with SO₂ exposure (0 to 5.5 microliters per liter for 16 hours), SO₂ absorbed, and the amount of visible injury sustained by the leaf tissues. Ethane production is correlated positively with the injury resulting from treatment with bisulfite ion. The rate of emission of ethane from leaf discs of cucurbit cultivars as a result of exposure to bisulfite solutions is in agreement with the order and the degree of their resistance to injury by SO₂. Thus, exposure to bisulfite and the subsequent release of ethane can be used to determine the relative resistance of different species and cultivars to SO₂ gas.     

Effect of nitrogen starvation on the level of adenosine 3',5'-monophosphate in Anabaena variabilis.

Low levels of adenosine 3',5'-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0--2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3--4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development.     

EMILIN-1 deficiency induces elastogenesis and vascular cell defects

EMILINs constitute a family of genes of the extracellular matrix with high structural similarity. Four genes have been identified so far in human and mouse. To gain insight into the function of this gene family, EMILIN-1 has been inactivated in the mouse by gene targeting. The homozygous animals were fertile and did not show obvious abnormalities. However, histological and ultrastructural examination revealed alterations of elastic fibers in aorta and skin. Formation of elastic fibers by mutant embryonic fibroblasts in culture was also abnormal. Additional alterations were observed in cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae. Considering that EMILIN-1 is adhesive for cells and that the protein binds to elastin and fibulin-5, EMILIN-1 may regulate elastogenesis and vascular cell maintenance by stabilizing molecular interactions between elastic fiber components and by endowing elastic fibers with specific cell adhesion properties.     

The importance of herbivory by protists in lakes of a tropical floodplain system

Inland aquatic ecosystems play a critical role in the global carbon cycle, processing a great fraction of the organic matter coming from terrestrial ecosystems, and the microbial food web is crucial in this process. Thus, we aimed to evaluate whether the food resource of planktonic protozoa comes mainly from small primary producers or heterotrophic bacteria in tropical shallows lakes, assuming the hypothesis that, in general, picocyanobacteria would be the main food resource for protists. We also expected that the autotrophic fraction would be mainly related to protists at the surface of the environments, while the heterotrophic fraction would be more important at the lower strata of the water column. We performed size-fractionation experiments to evaluate the effects of predation of protists on heterotrophic bacteria and picocyanobacteria. We also sampled planktonic organisms at the subsurface and bottom of 20 lakes in a Neotropical floodplain. We found an herbivory preference of heterotrophic flagellates, while ciliates seem to exert a stronger impact on heterotrophic bacteria. We also found no relationship between heterotrophic bacteria and protists in the field data, whereas positive relationships between picocyanobacteria and protists were observed in environments where there was sunlight. Thus, both heterotrophic bacteria and picocyanobacteria were important components in the food webs of tropical shallow lakes. Moreover, the trophic cascade caused by zooplankton predation suggests that protists are efficient in transferring the energy from the base of microbial food webs to higher trophic levels.