Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis.

The intracellular location of guanylate cyclase was examined in sperm from two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus, and from the tube worm Chaetopterus variopedatus. Cells suspended in a medium isotonic with sea water were passed repeatedly through a 23-gauge hypodermic needle to break flagella from heads. This preparation was then fractionated by two methods, one based on centrifugation over a 25% sucrose medium and the other involving repeated differential centrifugation, to resolve flagella from heads. Guanylate cyclase specific activity was increased 3.5–4.5-fold in the flagellar fraction relative to the starting sperm homogenate. Relatively little activity was present in the head fraction where specific activity was $\text{1}\text{10}\text{–}\text{1}\text{100}$ that of the flagella. Plasma membranes were separated from axonemal microtubules by dialyzing flagella against low ionic strength buffer, followed by centrifugation over a 40% sucrose medium. Although the overall recovery of guanylate cyclase was low, the specific activity in the plasma membrane fraction was increased two- to threefold over the dialyzed flagella, and over 90% of the recovered activity resided in this fraction. Thus the flagellar plasma membrane is a site rich in guanylate cyclase. It could not be determined, however, whether this is the only intracellular locale of the enzyme.     

Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase.

A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.     

Carbon dioxide assimilation in blue-green algae: initial studies on the structure of ribulose 1,5-bisphosphate carboxylase.

D-Ribulose 1,5-bisphosphate carboxylase was purified from the blue-green alga Anabaena cylindrica (Lemm) by procedures involving acid precipitation, ammonium sulfate fractionation, and Sephadex G-200 gel filtration. The enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and was a multimer of a single-size polypeptide chain of 54,000 daltons. The carboxylases from four species of blue-green algae (Anabaena, Nostoc strain MAC, Agmenellum quadruplicatum strain PR-6, and Anacystis nidulans strain TX20) were closely similar in molecular size, since enzyme activity was eluted at the same volume after sucrose gradient centrifugation. Further analysis by gel filtration indicated that the four blue-green algal carboxylases were nearly identical in molecular weight, ranging from 449 to 453,000. The amino acid composition of the Anabaena carboxylase was determined and was found to resemble closely the composition of the large subunit from eukaryotic photosynthetic organisms.     

CHARACTERIZATION OF PIGMENT MUTANTS IN A BLUEGREEN ALGA,ANACYSTIS NIDULANS1

Characteristics of 2 types of pigment mutants of the bluegreen alga, Anacystis nidulans, are described. “Yellow-green” mutants (YG) which have normal chlorophyll but only half the phycocyanin of the parent are similar to the parent in number of reaction centers/cell, number of chlorophylls/reaction center, maximum turnover rate of the reaction centers, quantum yields at 620 and 686 nm and specific growth rate; they have a reduced action at 620 nm. “Blue” mutants (BL) with somewhat higher phycocyanin but only one-third the chlorophyll of the parent are dissimilar to the parent. BL's have fewer reaction centers/cell, a smaller number of chlorophylls/reaction center, a higher maximum turnover rate of reaction centers, and a lower specific growth rate. BL's show ca. half the quantum yield of the parent at 620 nm and at 686 nm show a “red rise” rather than a “red drop.” The consequences attending low chlorphyll in the BL's are more drastic than those attending the low phycocyanin of the YG's.     

Biodegradation of Dinoseb (2-sec-Butyl-4,6-Dinitrophenol) in Several Idaho Soils with Various Dinoseb Exposure Histories

We examined the ability of native microorganisms in various Idaho soils to degrade dinoseb and studied some physical and chemical soil characteristics which might affect the biodegradation process. Dinoseb biodegradation rates were higher in silt-loam soils than in loamy-sand soils. Biodegradation rates were not influenced by previous exposure of the soils to dinoseb. Bacterial numbers, measured by standard plate counts on soil extract agar, were the best predictors of biodegradation rates, accounting for 53% of the variability between soils. Soil nitrate-N inhibited dinoseb biodegradation and accounted for 39% of the variability. Sorption of dinoseb to soil surfaces also appeared to influence biodegradation rates. No other soil parameter contributed significantly to the variability in biodegradation rates. Persistence of dinoseb in one soil was due to inhibition of biodegradation by nitrate, while in another soil persistence appeared to be due to lack of native degradative microorganisms.     

Interleukin 6 is essential for antibody secretion by human in vivo antigen-induced lymphoblastoid B cells

In vivo immunization of normal subjects with a variety of antigens generates circulating lymphoblastoid (LB) B cells, which in vitro spontaneously secrete significant levels of specific antibody. Since activation and initial differentiation of these cells occurs in vivo, they provide a useful model for the study of the later stages of B cell maturation. In the present study, we investigated the requirement of interleukin 6 (IL-6) for the "spontaneous" in vitro production of IgG-Tet by LB B cells. Addition of IL-6 to cultures of LB B cells in medium supplemented with 10% fetal calf serum failed to increase the levels of IgG-Tet produced in vitro. However, addition of anti-IL-6 antibodies decreased IgG-Tet production as much as 70%, and this inhibition could be reversed by the addition of IL-6. LB B cells cultured in serum-free medium in order to restrict endogenous IL-6 production secreted only low levels of antibody, unless exogenous IL-6 was added. Addition of 2.5 units/ml of IL-6 to serum-free cultures induced an increase in IgG-Tet secretion nearly comparable to that seen in cultures supplied with serum. The magnitude of the increase in IgG-Tet secretion in response to exogenous IL-6 was inversely related to the number of cells in culture, which was due in part to increased endogenous IL-6 production in cultures with higher cell concentrations. Experiments including hydroxyurea in serum-free cultures indicated that IL-6-dependent enhancement of LB B cells' IgG-Tet secretion was not primarily mediated by cell growth. These observations suggest that in vivo generated LB B cells are not totally committed to antibody secretion, and that IL-6 is essential for in vivo antigen-induced LB B cells to reach the antibody-secreting stage.     

Hypersensitivity pneumonitis in nonhuman primates. I. Studies on the relationship of immunoregulation and disease activity

We investigated the relationship of immunoregulation to disease activity in a nonhuman primate model of pigeon breeder's disease. Two Macaca arctoides monkeys developed classical symptoms of hypersensitivity pneumonitis after sensitization and prolonged bronchial challenge, whereas 2 other monkeys remained asymptomatic after in vivo challenge. There were no differences in the percentages of T cells, B cells, monocytes, or FC gamma-bearing T cells between symptomatic and asymptomatic animals. Nonetheless, we found a population of concanavalin A-induced, pigeon serum- (PS) induced, and spontaneous T cells that functioned as suppressor cells in autologous in vitro co-cultures in asymptomatic animals that were missing or nonfunctional in symptomatic animals. Monocyte suppressors functioned in both groups. We used low-dose total body irradiation (TBI) to inactivate T suppressor cells. Fifteen radiation units of TBI caused no change in the physical activity, routine chemistries, or blood counts of the 4 animals. After TBI, however, the previously asymptomatic animals developed fever, tachypnea, and signs of pulmonary congestion after in vivo challenge with PS. There was no change in the response to challenge in the symptomatic group. This altered response to in vivo challenge in the previously asymptomatic group persisted for 2 wk after TBI. During this period the difference in vitro immunoregulatory activity between Con A-induced, PS-induced, and spontaneous T cells in symptomatic and asymptomatic animals disappeared. Monocyte suppressors, however, continued to function in both groups after TBI. These data suggest that the monkey is an appropriate model for studies of human HP and that T cell immunoregulation may be an important element in the pathogenesis and disease activity of HP.     

Detection of glycogen in a glycogen storage disease by C nuclear magnetic resonance

The livers of gsd/gsd rats homozygous for the glycogen storage disease phosphorylase b kinase deficiency were observed by ¹³C NMR using a surface coil. Clear signals were detected from glycogen. The concentration of glycogen as determined by NMR was 3-times that found in normal strains agreeing well with chemical determinations Starvation did not significantly reduce the glycogen content of the livers with glycogen storage disease whereas it reduced the signal below detectability in normal rats. Difference spectra of starved normal rats from fed gsd/gsd rats gave spectra similar in appearance to that of purified glycogen. Glycogen both in vivo and in vitro is fully visible using ¹³C NMR.