Statistical Mechanics of Zooplankton

Statistical mechanics provides the link between microscopic properties of many-particle systems and macroscopic properties such as pressure and temperature. Observations of similar “microscopic” quantities exist for the motion of zooplankton, as well as many species of other social animals. Herein, we propose to take average squared velocities as the definition of the “ecological temperature” of a population under different conditions on nutrients, light, oxygen and others. We test the usefulness of this definition on observations of the crustacean zooplankton Daphnia pulicaria. In one set of experiments, D. pulicaria is infested with the pathogen Vibrio cholerae, the causative agent of cholera. We find that infested D. pulicaria under light exposure have a significantly greater ecological temperature, which puts them at a greater risk of detection by visual predators. In a second set of experiments, we observe D. pulicaria in cold and warm water, and in darkness and under light exposure. Overall, our ecological temperature is a good discriminator of the crustacean’s swimming behavior.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1)

Approximately 40 amino-terminal residues and 20 internal residues of CSF-1 purified from the media of cultured human pancreatic carcinoma (MIA PaCa) cells and of cultured murine L cells have been identified. Results indicated that the two subunits in each molecule of biologically active CSF-1 are identical in their amino-terminal portions. The twelve amino-terminal residues of MIA PaCa CSF-1 were found to be identical to those of human-urinary CSF-1, suggesting that the polypeptide portions of the two human proteins may be identical. Approximately 75% of the amino acids identified in both MIA PaCa CSF-1 and murine CSF-1 were found to be common to both. No homology to other proteins was observed. This study suggests a subunit polypeptide Mr nearer to 17K than to 26K predicted from cDNA.     

Purification of an antitrypanosomal factor fromPseudomonas fluorescens by high-pressure liquid chromatography

Studies on the purification of an antitrypanosomal factor (ATF-II) obtained fromPseudomonas fluorescens disclosed that high-pressure liquid chromatography (HPLC) with a Rad-pak porasil (10 silica) column and a GPC-60 Å column was an efficient procedure for the separation of the active components. Extraction of the factor with absolute ethanol prior to elution significantly enhanced the lytic activity of the HPLC eluates, as shown by marked pathologic changes followed by lysis in bioassays performed withTrypanosoma equiperdum. HPLC provided an increase of purification 30 times that obtained with gel filtration of the crude bacterial product. The lipopolysaccharide content of the purified fractions was markedly reduced and indicated an additional advantage for further in vivo tests in experimental infections withT. cruzi.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Studies with 6 beta-bromoacetoxyprogesterone

Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, copurified from the cytosol fraction of human term placenta, were identically inactivated by 6 beta-bromoacetoxyprogesterone. This affinity alkylating steroid binds at the enzyme-active site (Km = 866 microM; Vmax = 0.073 mumol/min/mg). Enzyme inactivation by four concentrations of 6 beta-bromoacetoxyprogesterone (molar ratio of steroid to enzyme, 71/1 to 287/1) causes irreversible and time-dependent loss of both the 17 beta- and 20 alpha-activities according to first order kinetics and affirms that the alkylating steroid is an active site-directed inhibitor (KI = 2.7 X 10(-3) M; k3 = 1.6 X 10(-3) s-1). Affinity radioalkylation studies using 6 beta-[2'-14C]bromoacetoxyprogesterone indicate that 2 mol of steroid are bound to each mole of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 6 beta-bromoacetoxyprogesterone carboxymethylates cysteine (56%), histidine (22%), and lysine (8%) residues in the active site. These results are identical with those reported for 2-bromo[2'-14C]acetamidoestrone methyl ether radioalkylation of purified "17 beta-estradiol dehydrogenase." The parallel inactivation of 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase by 6 beta-bromoacetoxyprogesterone further shows that both activities reside at a single enzyme-active site. The radioalkylation profile supports our proposed model of one enzyme-active site wherein the bound progestin and estrogen substrates are inverted, one relative to the other.     

Motion behavior of nauplii and early copepodid stages of marine planktonic copepods

The goal of this study was to quantify periods of activity andvelocities of late naupliar and early copepodid stages of planktoniccopepods occurring regularly on the southeastern continentalshelf of the USA. We obtained quantitative information on eightspecies, including adult females of Oithona plumifera. All studieswere conducted at food concentrations near or above satiationlevels. Activities ranged from 0.85% (adult females of O.plumifera)to 100% of time (nauplii and copepodids of various calanoidspecies). Motion velocities (excluding escape motion) coveredmore than one order of magnitude: from 0.39 mm s^–1 fornauplii of Temora stylifera to 5.24 mm s^–1 for naupliiof Oncaea mediterranea. Ranges of activities of species rangefrom occasional for early juveniles to adult females of O.plumiferato 100% for the same range of T.stylifera, the latter creatinga feeding current from N III onwards, the former not at all.Of notable interest is Centropages velificatus which moves intermittentlyas a late nauplius, continuously as an early copepodid and intermittentlyas an adult. All observed calanoid late nauplii and copepodidsmove in three dimensions, excluding copepodids of the shelfbreak/oceanicParacalanus aculeatus. The results indicate not only significantdifferences in motion behavior between cyclopoids and calanoids,but also between calanoid species. Yet, some calanoid speciesshow little ontogenetic changes at all.     

The novel flightless-I gene brings together two gene families, actin- binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction

The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues. In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin- binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions. Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin- associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup. Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins. There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction. It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins.      

Estradiol 17β-dehydrogenase and 20α-hydroxysteroid dehydrogenase from human placental cytosol: one enzyme with two activities?

The soluble enzyme estradiol 17β-dehydrogenase (17β-ED) from human term placental cytosol is reported to be a stereospecific oxidoreductase for estrogen substrates. A published purification scheme (heat treatment and affinity chromatography) yielded a homogeneous protein which had the reported characteristics of pure 17β-ED and also had 20α-hydroxysteroid dehydrogenase (20α-HSD) activity. Spectrophotometric assay when the buffer contained albumin, 8 mg/ml, masked the 20α-HSD activity observed in albumin-free conditions and may explain why this bifunctional activity has gone unrecognized. In human placenta, one enzyme may catalyze stereospecific oxidation/reduction of both estrogen and progesterone.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.