Increases in arterial blood oxygen during exercise in the lemon shark (Negaprion brevirostris)

Summary Polyethylene cannulae were implanted in pre- and post-branchial blood vessels allowing nonstressful blood sampling over a variety of activity ranges in an active tropical elasmobranch, the lemon shark (Negaprion brevirostris). TheP ₅₀ was found to be 11.8 Torr at 24°C and pH of 7.7. A Bohr shift of –0.36 was also found. BloodP _{o 2} and oxygen content were measured during rest, routine swimming, and exercise in unanesthetized, free swimming juveniles. Under all conditions venous oxygen levels were low with venousP _{o 2} of 7.1±2.7 Torr, and venous oxygen content ( ) of 0.56±0.4 vol%. However, a large variability was found in arterial blood measurements. ArterialP _{o 2} ranged from 7 to 80 Torr, while arterial oxygen content (Ca_{o 2}) varied from 1.6 vol% to 6.8 vol% among ten experimental animals. A significant increase in arterialP _{o 2}, oxygen content, and hematocrit was noted during increased activity. Since the venous system provides little or no oxygen reserve, increased oxygen extraction from the blood ( ) appears to be met by an increase inCa_{o 2} rather than a decrease in . Mechanisms to accomplish this may include increasing hematocrit and vacular gill shunts.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. I. Effect of size.

Aspects of protein structure determining endocytosis of proteins by sinusoidal rat liver cells in vivo have been studied, using cross-linked or aggregated derivatives of bovine pancreatic ribonuclease A (labelled with 125I) as probes. Ribonuclease was cross-linked by reaction with dimethylsuberimidate, a way of modification that does not change the charge of the protein. Monomer, dimer and polymer fractions were isolated by gel filtration and characterized in respect of size and number of amino groups modified. Maintenance of enzyme activity, stability of disulfide bonds, and lack of susceptibility to endoproteases showed that the cross-linking procedure did not result in gross conformational changes of the ribonuclease molecules. Monomer, dimer and polymer fractions were injected into nephrectomized rats and plasma clearance and uptake in liver and spleen were determined. About 30% of the injected polymer fraction was found in liver 15 min after injection; for dimer and monomer fractions values of 6% and 2% of the dose were found. Similar differences were found in spleen. Autoradiography, cell isolation, and subcellular fractionation showed that in liver the radioactive proteins were taken up in lysosomes of sinusoidal cells. Similar results were obtained with fractions of aggregated ribonuclease prepared by freeze-drying the protein from 50% acetic acid. Our results demonstrate that the rate of uptake of the ribonuclease derivatives is positively correlated with the size of the molecules. Similarity of the results obtained with cross-linked and aggregated fractions suggests that the number of ribonuclease 'subunits'/molecule, rather than the procedures used to prepare the polymers, determine the rate of uptake by liver and spleen.     

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. II. Effect of charge.

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic groups on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways. Firstly, we changed the positively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolished their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polymer and 32% of the succinylated polymer were endocytosed by liver after 6 h. For the dimer fractions these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polymer in the lysosomal/microsomal fraction of sinusoidal liver cells, probably endothelial cells. Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives to the cation exchanger SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.     

Bioaccumulation of silver by a multispecies community of bacteria

A stable community of bacteria that had unusually high tolerance of soluble silver was isolated from soil by chemostat enrichment. The community consisted of three bacteria: Pseudomonas maltophilia, Staphylococcus aureus and a coryneform organism. The pseudomonas was primarly responsible for the silver resistance. The tolerance of high silver concentrations, up to 100 mM Ag⁺, was greatly reduced when the community was grown in the absence of silver. Pseudomonas maltophilia comprised approximately 50% by numbers of the community when grown in chemostats in the presence or absence of Ag⁺ but large fluctuations occurred in population sizes of the other two bacteria; the S. aureus population was small (less than 1%) in the presence of Ag⁺ but comparised a third of the total numbers when Ag⁺ was omitted from the medium. Silver-resistant respiration of the silveradapted community was significant even when it was confronted with high concentrations of Ag⁺. In contrast the respiration of the coryneform organism and particularly S. aureus was highly sensitive to silver. The inhibition constants for silver-sensitive respiration were 0.78 mM and 0.04 mM for silver acclimatized and nonacclimatized communities respectively.The community had great capacity for silver bioaccumulation. Maximum concentrations of over 300 mg silver per g dry weight of biomass were recorded at an accumulation rate of 21 mg Ag⁺ h^-1 (g biomass)^-1. The extent of silver removal from solution was a function of initial concentration of silver; at low external concentrations (ca. 1 mM) all the silver was rapidly removed from solution, at high concentrations (ca. 12 mM) 84% removal occurred in 15 h.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.