New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.     

Insulin-stimulated hexose transport and glucose oxidation in rat adipocytes is inhibited by sphingosine at a step after insulin binding

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.     

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst

The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.     

NADH dehydrogenase from bovine neutrophil membranes. Purification and properties

A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.     

Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.     

On the biological occurrence and regulation of 1-acyl and 1-O-alkyl-diradylglycerols in human neutrophils. Selective destruction of diacyl species using Rhizopus lipase

The occurrence and regulation of 1-ether-linked diradylglycerol in human neutrophils were investigated using a sensitive and practical analytical mass method which distinguishes 1-O-alkyl- (EAG) versus 1-acyl (DAG) diglycerides. After phosphorylation of diglycerides to the corresponding [32P]phosphatidic acids using [gamma-32P]ATP and diglyceride kinase (Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600), lipase from Rhizopus arrhizus selectively degraded the 1-acyl-containing species (DAG), but the ether lipid (EAG) was resistant and was identified and quantified after thin layer chromatography separation. By using this method, unstimulated neutrophils were demonstrated to contain both DAG and EAG (100-180 and 40-95 pmol/10(7) cells, respectively). The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) caused a rapid (30 s) and transient increase (1.6-fold) in DAG, but no increase in EAG. Opsonized zymosan produced a 6-8-fold sustained increase in DAG peaking at 2 to 3 min, but only a small (1.7-fold) increase in EAG which was not seen until later times (10 min). Thus, under these stimulation conditions, the major diglyceride was DAG. However, in neutrophils "primed" with cytochalasin B or phorbol ester, formyl-methionyl-leucyl-phenylalanine caused a significant increase in EAG. Neutrophils pretreated with cytochalasin B and then stimulated by fMLP showed a rapid (15-60 s) increase (more than 3-fold) in total diglycerides which was sustained beyond 5 min. At the earliest time points (15-30 s), the increase was due almost entirely to DAG (3-fold), but at 1 min and beyond, EAG comprised as much as 40% of the total (up to a 5-fold increase in EAG). Neutrophils pretreated with phorbol ester prior to fMLP stimulation showed a rapid (around 30 s) more than 2-fold increase in both DAG and EAG. Thus, priming conditions (in particular cytochalasin B) may alter either the access of phospholipase(s) C and/or D to membrane phospholipids or may affect their activities, allowing hydrolysis of 1-O-alkyl-containing lipids to generate 1-O-alkyl-containing diglycerides.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.