The phylogenetic odyssey of the erythrocyte. II. The early or invertebrate prototypes

Freely existing hemoglobin-bearing cells suspended in a plasmic milieu (erythrocytes) are found in a relatively small number of taxanomically scattered invertebrates. These species include some annelids, echiurids, molluscs, phoronids, nemerteans and echinoderms, e.g. Pista pacifica, Urechis caupo, Noetia ponderosa, Phoronis australis, Lineus fuscoviridis and Cucumaria miniata respectively. The typical invertebrate erythrocyte (hemocyte, coelomocyte) can be described as permanently nucleated, considerably larger than the human red cell, oval or circular in configuration and spherical, biconvex or flattened in profile. The marginal band of the erythrocyte, a bundle of subplasmalemmal microtubules that circumscribes the periphery of the cell and lies in the plane parallel to its flat surface makes its first appearance in certain invertebrates. This structure in association with the cell surface-associated cytoskeleton is responsible for the flattened elliptical shape seen in some invertebrate erythrocytes and endows them with flexibility and resilience to mechanical forces. This in an evolutionarily persistent characteristic that is retained throughout the submammalian vertebrates. The erythrocytes of invertebrates are more morphologically and functionally diversified than the mammalian model. In addition to respiratory activities (oxygen storage and transport) they can sometimes function as vendors of nutrients and participate in other less obvious processes. These cells therefore frequently not only retain organelles that are usually discarded by vertebrate erythrocytes (ribosomes, golgi apparatus, etc.) but may also depending upon the species, manifest in their cytoplasm organelles and inclusions that are not a normal component of developing or mature submammalian vertebrate and mammalian erythroid cells. Examples of the latter are pigment granules, lipid droplets, extensive glycogen stores and prominent Prussian blue positive inclusions. Erythrocytes in the invertebrates, though presenting certain cytologic and functional features in common, are a heterogenous collection of cells, each tailored for a specific species or group of organisms.     

A comparative study of animal erythrocyte agglutinins from marine algae

1. Fifteen marine algal species were analyzed for agglutinins to rabbit, sheep and human A, B and O blood group erythrocytes. 2. Protein extracts from all marine algae agglutinated rabbit erythrocytes, whereas twelve and five extracts agglutinated sheep and human erythrocytes, respectively. 3. The highest agglutination titers were consistently observed with rabbit erythrocytes. 4. Dictyota dichotoma strongly agglutinated human B blood group erythrocytes relative to A and O group erythrocytes. 5. Agglutination titer of rabbit erythrocytes by six algal extracts was not inhibited by mono- or polysaccharides, yet was reduced by glycoproteins.     

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species.

Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a Km for NG of 52 +/- 4 microM, a Km for NADPH of 28 +/- 2 microM, and a Vmax of 124 +/- 6 microM x min(-1), while the P. fluorescens enzyme had a Km for NG of 110 +/- 10 microM, a Km for NADPH of 5 +/- 1 microM, and a Vmax of 110 +/- 11 microM x min(-1). Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an approximately 2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions or middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.     

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.     

The invertebrate population and response to pesticide treatment of two permanent and two temporary pastures

During 1979 and 1980 the herbage yields of two permanent pastures and two temporary swards were compared. All four swards received 250 kg N/ha per yr. The invertebrate population of all four swards was studied. Pot-worms (Enchytraeidae) and some species with long life cycles, e.g. wireworms (Agriotes spp.) were more numerous in the permanent swards, but aerial species were more numerous in the temporary swards. A range of pesticide treatments was applied. At one temporary sward site, application of the broad-spectrum pesticide aldicarb increased total annual yield of herbage by 16% in 1979 and 33% in 1980. Insecticide application at the same site resulted in no increase in herbage yield in 1979 and 12% yield increase in 1980. At the other three sites no significant increases in total annual yield were recorded in either year, but there were significant responses at one harvest or more at every site.     

Isolation and partial characterization of two minor glycoproteins from human erythrocyte membranes

Two minor glycoproteins GP-II and GP-III, were isolated from human erythrocyte membranes and characterized chemically and immunologically. The chemical composition of GP-II and GP-III was similar: GP-II consisted of 81% protein and 19 % carbohydrate of which 4.9 % was hexose. 5.4 % hexosamine and 7.8 % sialic acid. GP-III consisted of 76 % protein and 24 % carbohydrate of which 7.6 % was hexose, 7.2 % hexosamine and 8.1 % sialic acid. The amino acid composition of GP-II and GP-III was also similar. GP-II and GP-III, however, differed in chemical composition from the MN glycoprotein. GP-II and GP-III were associated with the blood group activities Ss, I and A, but not with the MN antigens. GP-III had higher blood group activities per μg of protein than did GP-II. The specific activities for the Ss blood group antigens were increased 3–10-fold by purification of GP-III from the aqueous phase of chloroform methanol extracts.     

The characterization of invertebrate troponin C

TNCs from lobster, mussel, and squid migrated with rabbit TNC at an apparent mol. wt of 18,000. Electrophoretic mobilities in the presence or absence of Ca2+ were compared: the electrophoretic mobility of rabbit TNC was greater in the presence of Ca2+ than it its absence, but all invertebrate TNCs tested migrated identically, whether Ca2+ was present or not. The Ca2+-binding capacity of invertebrate TNCs was only one Ca2+ ion per molecule. The alpha-helix contents in the presence or absence of Ca2+ were compared: rabbit TNC changed by a value of 16% and invertebrate TNCs by 4%. Antibodies to loligo TNC did not cross-react with rabbit TNC, but did interact with their molluscan TNCs.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Presence of translational inhibitory activity in partially purified extracts from two Petrocoptis species

The presence of translational inhibitory activity in partially purified extracts from several paleoendemisms from Spain was investigated. The precipitates from 40-80% (NH4)2SO4 fraction from Petrocoptis glaucifolia and Petrocoptis grandiflora displayed a strong inhibitory activity on the protein synthesis of cell-free extracts from rat liver, rabbit reticulocytes and yeast and to a much lower extent on the protein synthesis in isolated rat liver cells. The inhibitors seem to be proteins since they were precipitated by high salt concentrations, were non-dialysable and were inactivated by heat. Since the partially purified extracts did not show unspecific RNA-A or protease activities, the active compounds can be considered to belong to the plant ribosome-inactivating proteins.     

Agricultural disturbance response models for invertebrate and algal metrics from streams at two spatial scales within the U.S.

As part of the USGS study of nutrient enrichment of streams in agricultural regions throughout the United States, about 30 sites within each of eight study areas were selected to capture a gradient of nutrient conditions. The objective was to develop watershed disturbance predictive models for macroinvertebrate and algal metrics at national and three regional landscape scales to obtain a better understanding of important explanatory variables. Explanatory variables in models were generated from landscape data, habitat, and chemistry. Instream nutrient concentration and variables assessing the amount of disturbance to the riparian zone (e.g., percent row crops or percent agriculture) were selected as most important explanatory variable in almost all boosted regression tree models regardless of landscape scale or assemblage. Frequently, TN and TP concentration and riparian agricultural land use variables showed a threshold type response at relatively low values to biotic metrics modeled. Some measure of habitat condition was also commonly selected in the final invertebrate models, though the variable(s) varied across regions. Results suggest national models tended to account for more general landscape/climate differences, while regional models incorporated both broad landscape scale and more specific local-scale variables.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.