Immune complex effects on murine macrophages. I. Immune complexes suppress interferon-gamma induction of Ia expression

We have studied the effects of immune complexes on the expression of macrophage surface proteins in vitro. Increased expression of the H-2 molecules I-A, I-E, and K on the macrophage membrane was induced by in vitro culture with crude lymphokine or interferon-gamma. Expression of all three of the molecules was additionally increased by stimulating the cultures with heat-killed Listeria monocytogenes. Addition of soluble immune complexes to the cultures did not have any effect on macrophage expression of these proteins. However, significant inhibition of lymphokine or interferon-gamma induction of I-A, I-E, and H-2K was observed when macrophages were cultured on plates to which immune complexes had been bound. This inhibition was dose dependent, required an immunoglobulin (Ig) molecule with an intact Fc portion, did not require the presence of T cells, and occurred in the presence of indomethacin. Complexes containing IgG1, IgG2a, IgG2b, and IgE, but not IgM or IgA, antibodies mediated the inhibitory effect.     

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.     

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

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Haemoglobins of invertebrate tissues. Nerve haemoglobins of Aphrodite, Aplysia and Halosydna

1. The occurrence of haemoglobin in invertebrate nerves is surveyed. Haemoglobin was observed in the nerves and ganglia of the marine nematode Amphiporus sp. and of the polychaet annelid Halosydna sp. 2. Haemoglobins from the nerve and ganglia of the polychaet annelid Aphrodite aculeata L. and from the nerve of the gastropod mollusc Aplysia californica have been partially purified. The haem in each case was identified as iron protoporphyrin IX. 3. The minimum molecular weight of Aphrodite nerve haemoglobin deduced from the haem content and amino acid analysis is 17090, in agreement with the molecular weight 15600+/-1000 determined by sedimentation equilibrium. 4. The molecular weight of Aplysia nerve haemoglobin was determined by sedimentation equilibrium to be 16400+/-1000. 5. The oxygen dissociation curves are hyperbolic. Half-saturation is achieved at 1.1mm. Hg for Aphrodite nerve haemoglobin and at 4.0mm. Hg for Aplysia nerve haemoglobin. The coefficients for partition between carbon monoxide and oxygen are: Aphrodite nerve haemoglobin, 167; Aplysia nerve haemoglobin, 116. 6. The ferrous haemoglobins combine with cyanide. 7. We conclude that the intracellular haemoglobins of muscle and nerve are similar.     

Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++

Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.