Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

Sizes and mass distributions of clathrin-coated vesicles from bovine brain

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.     

Interactions of erythrosin B (U.S. F,D & C Red 3) with rat cortical membranes

Erythrosin B inhibits Na, K-ATPase in rat brain tissue as demonstrated by studying glycoside binding, ATPase activity and ion fluxes. The potency of the noncompetitive inhibition of [³H]-ouabain binding by erythrosin B is influenced by glycoside concentration, monovalent cation concentration, and incubation time. [¹⁴C]- Erythrosin B binds to synaptic membranes prepared from rat cortex. Erythrosin B and some of its structural analogs inhibit both [³H]-ouabain and [¹⁴C]-erythrosin B binding, but ouabain and other glycosides do not inhibit the binding of [¹⁴C]-erythrosin B. Subcellular distributions of [³H]-ouabain and [¹⁴C]- erythrosin B binding in fractionated cortical tissue preparations are equivalent and parallel ATPase activity. The dissimilar response of [³H]-ouabain binding and [¹⁴C]-erythrosin B binding to changes in tissue preparation, incubation temperature, and partial solubilization of binding sites by deoxycholate (DOC) Suggests two separate binding sites for erythrosin and ouabain to rat cortical membranes.     

Apolipoprotein A-I isoprotein synthesis by the perfused rat liver

The hepatic pattern of synthesis of the apolipoprotein A-I isoforms has been analyzed in the rat. After isolated livers were perfused with defibrinated rat blood and [³H]leucine, the radioactivity associated with apolipoprotein A-I and other apolipoproteins was determined following two-dimensional gel electrophoresis of the perfusate d < 1.21 g/ml lipoprotein fraction. In rat serum, apolipoprotein A-I is a polymorphic system consisting of two major isoproteins and a series of minor species. Following liver perfusion, 72% of the radioactivity associated with apolipoprotein A-I isoproteins was recovered in the more acidic and quantitatively less abundant of the two major isoforms. Only 8% was associated with the major apolipoprotein A-I isoform, and similar or lower amounts were found in the other minor isoproteins. These results are consistent with the concept that, in the rat, the major apolipoprotein A-I isoforms differ in their pattern of biosynthesis and/or metabolism.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Calcium transport in dispersed acinar cells from rat pancreas

The present studies were performed to attempt to elucidate the basis for the discrepancy between results of Kondo and Schulz (1976, Biochim. Biophys Acta 419, 76–92), who found that cholecystokinin and cholinergic agents increase uptake of ⁴⁵Ca by dispersed acinar cells from rat pancreas, and results of others (Matthews, E.K., Petersen, O.H. and Williams, J.A. (1973) J. Physiol. 234, 689–701; Chandler, D.E. and Williams, J.A. (1974) J. Physiol. 243, 831–846; Case, R.M. and Clausen, T. (1973) J. Physiol. 235, 75–102; Gardner, J.D., Conlon, T.P., Klaeveman, H.L., Adams, T.D. and Ondetti, M.A. (1975) J. Clin. Invest. 56, 366–375; Christophe, J.P., Frandsen, E.K., Conlon, T.P., Krishna, G. and Gardner, J.D. (1976) J. Biol. Chem. 251, 4640–4645; Shelby, H.T., Gross, L.P., Lichty, P. and Gardner, J.D. (1976) J. Clin. Invest. 58, 1482–1493 and Deschodt-Lanckman, M., Robberecht, P., de Neef, P., Lammens, M. and Christophe, J. (1976) J. Clin. Invest. 58, 891–898). They have reported that cholecystokinin and cholinergic agents do not alter or cause a slight decrease in uptake of ⁴⁵Ca by pancreatic acinar cells. Our present results indicate that increased uptake of ⁴⁵Ca by acinar cells incubated with cholecystokinin occurs only in cells washed with iced, 160 mM choline chloride and reflects increased cellular uptake of radioactivity from the wash solution but not from the incubation medium. We detected no effect of cholecystokinin on uptake of ⁴⁵Ca by cells washed with 160 mM choline chloride containing 5 mM ethylenediaminetetraacetate or by cells washed with Krebs-Ringer bicarbonate. Furthermore, cells washed with 160 mM choline chloride accumulated a substantial amount of ⁴⁵Ca from the wash solution and this accumulation was increased in cells that had been preincubated with cholecystokinin. Cells washed with Krebs-Ringer bicarbonate did not take up ⁴⁵Ca from the wash solution.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Activation of macrophage adenylate cyclase by stimulants of the oxidative burst and by arachidonic acid--two distinct mechanisms

The effect of agents stimulating the oxidative burst (OB) in oil-elicited guinea pig peritoneal macrophages (MPs) on cyclic adenosine 3′,5′-monophosphate (cAMP) levels was examined. We found that: (i) Phorbol myristate acetate (PMA), the Ca²⁺ ionophore A23187, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and opsonized zymosan, elevated cAMP levels two- to fivefold; (ii) the biologically inactive PMA analog, 4-O-methyl-PMA, was proportionally less effective than PMA in stimulating cAMP accumulation; (iii) increased levels of cAMP were evident after 10 min of incubation with the stimulants, in the presence of the phosphodiesterase inhibitor 3-isobutyl methylxanthine (IBX); (iv) basal cAMP levels in MPs increased proportionally with the extracellular Ca²⁺ concentration; (v) the cAMP-elevating effect of all stimulants (with the exception of A23187) was more pronounced in low Ca²⁺ media, associated with lower basal cAMP levels. A23187 did not elevate cAMP levels in the absence of extracellular Ca²⁺; (vi) short-term incubation of MPs with arachidonic acid and with the arachidonic acid precursor, linoleic acid, induced an increase in the level of cAMP; (vii) the elevations in cAMP levels induced by OB stimulants were enhanced, not blocked, by mepacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin or aspirin, demonstrating that prostaglandin (PG) synthesis was not involved; (viii) the cAMP-elevating effect of arachidonic and linoleic acids was blocked by ETYA and indomethacin, indicating that it was mediated by PGs. The mechanism by which OB stimulants elevate cAMP levels could not be determined but changes in the cellular level of Ca²⁺ seem to play a pivotal role.     

Rat leukocyte inhibitory factor (LIF): Similarities to human LIF and generation by cells from rats with collagen-induced arthritis

Rat lymph node cells (LNC) produce a mediator which exhibits functional and physicochemical similarities to human leukocyte inhibitory factor (LIF). Measurement of LIF can be used to quantify cellular sensitivity in rats to foreign protein antigens or to a heterologous antigen in vitro. Concanavalin A-induced rat LIF has a molecular weight of 100,000-60,000 daltons and retains activity after heating to 56 °C for 30 min. Rat LIF is not synthesized in the presence of puromycin and appears to be a protein, since it is inactivated by treatment with chymotrypsin. Moreover, the activity of rat LIF is susceptible to the serine esterase inhibitor, diisopropylphosphofluoridate, but it is resistant to neuraminidase treatment. Finally, rat LIF preferentially inhibits the migration of rat and human polymorphonuclear leukocytes but not that of rat or guinea pig peritoneal exudate cells enriched for macrophages. Except for the more dispersed size of rat LIF, these properties are analogous to those described for human LIF. LIF activity is generated, in an antigen-specific manner, by LNC sensitized to ovalbumin or purified protein derivative of tuberculin when cultured with the antigen used for sensitization. LNC from rats rendered arthritic by prior intradermal (id) injection of native chick type II collagen in incomplete Freund's adjuvant produce LIF in response to this heterologous antigen. These studies delineate a new assay for cellular sensitivity in rats and provide additional evidence that cellular reactivity to type II collagen is present in this animal model of arthritis.