Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

Respiratory physiology and energy conservation efficiency of Campylobacter jejuni

A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.     

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

The parasitic coelenterate, Polypodium hydriforme USSOV, from the eggs of the American acipenseriform Polyodon spathula.

No significant differences in macro- and micromorphology were found between the parasitic stolon and free-living polyps of Polypodium sp. obtained from infected eggs of the North American acipenseriform fish Polyodon spathula and corresponding developmental stages of Polypodium hydriforme Ussov, parasitic in the Volga sterlet (Acipenser ruthenus). Therefore, both the American and the European forms of Polypodium belong to the species P. hydriforme Ussov.     

DNA-damaging and mutagenic effects of 1,2-dimethylhydrazine on Bacillus subtilis repair-deficient mutants.

Mutagenic, DNA-damaging, and in vivo alteration of DNA have been demonstrated for 1,2-dimethylhydrazine (DMH), a potent inducer of adenocarcinomas of the large intestine and colon of rats. These activities are pH-dependent, with 6.5 giving optimum response. There was no requirement for metabolic activation with rat-liver S9 mix when the appropriate Bacillus subtilis mutant strains were used. The Rec- strains recA8 and mc-1 were greater than 300-fold more sensitive to the DNA-damaging activity of DMH than was their isogenic wild-type parent. The DNA isolated from DMH-treated mc-1 had altered spectroscopic characteristics, and gave a greatly reduced transformation efficiency. Treatment of B. subtilis strain TKJ6321 with DMH at pH 6.5 induced His+, Met+ mutations in substantial numbers at low concentrations of this chemical. The use of B. subtilis mutants in these studies has therefore made it possible to demonstrate mutagenic and DNA-damaging activity in bacteria for this potent carcinogenic chemical.     

Adrenergic control of hypothalamic function during osmotic stress in the mallard duck (Anas platyrhynchos)

Summary The role of the paraventricular nucleus (PVN) and biogenic amines (BA) in regulating the level of corticoids in the serum of osmotically stressed mallard ducks (Anas platyrhynchos) was analyzed employing three experimental approaches: 1) pharmacologic alteration of central BA levels, 2) microscopic evaluation of BA distribution, and 3) placement of electrolytic lesions into the PVN. Reserpine and -methyl-p-tyrosine (mpt), agents that decrease the amount of BA's in the central nervous system, produced a fivefold increase in the concentration of serum corticoids. Conversely, pargyline and amphetamine, agents that increase the functional pool of BA's, prevented the rise in serum corticoid concentration normally observed in birds challenged with an intraperitoneal injection of hypertonic saline. When the topographic distribution of BA's was analyzed in the brains of osmotically stressed and nonstressed ducks distinct changes in the intensity of catecholamine (CA) fluorescence were observed in only one location, the PVN of the hypothalamus. Additionally, electrolytic lesions stereotaxically placed in the PVN blocked the osmotic stress-induced rise in serum corticoid concentration. These data therefore indicate that the PVN in the mallard duck plays some role in regulating the observed stress-induced rise in serum corticoid concentration, and that this regulatory function is probably inhibited by catecholamines.This research was supported by research grant No. GB 33321 from the National Science Foundation. We wish to express our sincere thanks to Mr. Howard Funk, research director, Colorado Division of Wildlife, for the use of the State's animal facilitiesThis research was submitted as partial fulfillment for the degree of Doctor of Philosophy, Department of Physiology and Biophysics, Colorado State University, Ft. Collins, CO 80521     

The preferential adsorption of hemoglobin to polyethylene.

Hemoglobin adsorption to foreign surfaces has not previously been considered in studies of blood-material interactions, despite the fact that hemoglobin is the most abundant protein present in blood. A hemoglobin-like protein was detected on a number of surfaces exposed to blood plasma, serum, and red cell suspensions. Hemoglobin adsorption to polyethylene from plasma was found to approximately equal the amount of adsorption of albumin and fibrinogen. The high relative affinity of hemoglobin for polyethylene was further confirmed by adsorption isotherm and direct competition experiments. The data from all four experimental methods support the following ranking of plasma protein affinity for polyethylene: Hemoglobin greater than fibrinogen greater than albumin congruent to gamma-globulin.     

Leukocyte migration in guinea pigs. II. Partial characterization of a leukocyte migration inhibitory factor distinct from macrophage migration inhibitory factor.

In this study we present data on the partial biological and biochemical characterization of guinea pig leukocyte migration inhibition factor (LIF) and migration inhibition factor (MIF). The results indicate that guinea pig LIF and MIF are distinct mediators of cellular immunity, in terms of indicator cells affected and molecular weight. This is in agreement with previous reports showing distinctions between human LIF and MIF. Partial characterization of guinea pig LIF suggested that it is a heat-stable protein of molecular weight 68,000–158,000 and does not contain terminal sialic acid groups.     

A new method for cell volume measurement based on volume exclusion of a fluorescent dye

Several methods currently in use for measuring mean corpuscular volume include: centrifuged packed cell volume, electronic impedance, and light scattering methods. Although these techniques are widely used and accepted, there are problems inherent to each method which may produce systematic errors that are difficult to estimate. This paper describes a new flow cytometric method of cell volume determination, based on the principle of volume exclusion, which may overcome the systematic errors of the methods currently in use. This method requires that the cells be suspended in a fluorescent dye which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence which is directly proportional to the cell's volume. The volume exclusion method is shown to give an estimate of mean red cell volume which correlates well with existing methods.