Cellular immunity in the mouse. V. Further studies on leukemia virus activation in allogeneic reactions of mice: stimulatory parameters.

The relationship between activation of thymic (T)-derived lymphocytes and mouse leukemia virus (MuLV) induction were studied in vivo and in vitro. The results indicate that there is no simple relationship between the severity of GVH, assayed by splenomegaly, alteration of T-cell reactivity in vitro, the activation of mouse leukemia viruses, and the subsequent development of lymphoma. Allogeneic stimulation either in vivo or in vitro is a potent activator of MuLV, as is the drug iododeoxyuridine. However, nonspecific T-cell mitogens such as PHA or Con-A, the drug cyclophosphamide, or specific antigenic stimulus such as sheep red blood cells after in vivo sensitization are not effective virus activators. The source of the cell supporting MuLV replication in vitro appears to be a theta-positive (T) lymphoblast.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

The reactions of the phosphorylated pathway of L-serine biosynthesis: thermodynamic relationships in rat liver in vivo

The effect of the induction of the enzymes of the phosphorylated pathway of L-serine biosynthesis on the thermodynamic relationships among the reactions has been determined in rat liver in vivo. The mass action ratios of the reactions involved were calculated from the concentrations of appropriate metabolites in freeze-clamped liver from animals fed normal and low-protein diets for 2 weeks. These ratios were compared with the equilibrium constants of the same reactions previously determined under physiological conditions and the results previously obtained in the rabbit. The thermodynamic relationships in the pathway were different between the normal rat and rabbit as might have been expected, because of the significantly lower activities of the L-serine biosynthetic enzymes in the former animal. Although the delta G for the overall pathway is nearly identical in the rat and rabbit (-5.8 versus -5.5 kcal/mol, respectively), the distribution of delta G among the reactions is different. The disequilibrium in the pathway in rat liver is nearly equally divided between the L-phosphoserine phosphatase (EC 3.1.3.3) step and the other two reactions [D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) and L-phosphoserine aminotransferase (EC 2.6.1.52)], whereas in rabbit the phosphatase reaction accounts for nearly the entire delta G. Feeding the rat a low protein diet, however, induced the activity of D-3-phosphoglycerate dehydrogenase 12-fold, that of L-phosphoserine aminotransferase 20-fold, and that of L-phosphoserine phosphatase 2-fold. With the induction of the pathway, L-phosphoserine appeared in the tissue, there was a more than 3-fold rise in L-serine in the liver, and the pattern of delta G in the rat liver approached that in the rabbit.     

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver.

The nicotinamide nucleotide dimers (NAD)2 and (NADP)2, obtained by electrochemical reduction of NAD+ and NADP+, are able to reduce such single-electron acceptors as the proteins cytochrome c, azurin and methaemoglobin, though at different rates. Under the same conditions the reduced nicotinamide coenzymes NADH and NADPH are not able to reduce these proteins at measurable rates unless a catalyst (phenazine methosulphate or NADH-cytochrome c reductase in the case of cytochrome) is present. The redox mechanism seems to involve the formation of an NAD(P). radical that in the presence of O2 gives rise to superoxide (O2.-), since superoxide dismutase inhibited these reactions.     

Studies on calcium binding to brush-border membranes from rabbit small intestine

A study was made of the uptake of Ca2+ by brush-border membrane vesicles prepared from rabbit small intestine. The process was found to be time, temperature and substrate concentration dependent, displayed saturability, did not depend on added energy sources and occurred optimally in a pH range of 7.5-8.0. Although the transport of D-glucose by these membrane vesicles responded to changes in osmotic pressure as modified by adding cellobiose to the medium, the uptake of Ca2+ was found not to be osmotically-sensitive. Moreover, the equilibrium uptake value obtained when vesicles were exposed to 0.36 mM Ca2+ was some 60-fold higher than the amount that could have been accommodated by the intravesicular space, calculated from the equilibrium uptake of D-glucose. It was concluded from these results that the uptake involved complete binding of the Ca2+ to the membrane. The ionophore A23187 enhanced the rates of uptake and efflux of Ca2+ without affecting equilibrium values, which suggests that the binding of Ca2+ measured under our conditions was to interior sites of the membrane. The binding capacity was decreased in the presence of 10 mM lidocaine as indicated by a diminution of the equilibrium binding values. Generating an electrochemical potential (negative inside) by addition of valinomycin to vesicles pre-equilibrated with K2SO4, enhanced the rate of uptake of Ca2+. Addition of metal ions, on the other hand, inhibited the uptake, La3+ and Tb3+ being most effective followed by Mn2+, Ba2+ and Mg2+. Na+ and K+ were the least inhibitory. The properties of the Ca2+ uptake process found in rabbit brush-border membranes were compared to those of similar processes occurring in other species.     

Differential effects of adenosine and verapamil on histamine vascular contractions

The present study was undertaken to compare the effects of adenosine and verapamil on histamine-induced contractions in rabbit vascular smooth muscle. Ring segments of rabbit femoral artery were isometrically mounted and contractile responses to histamine (10(-7) to 10(-4) M) were recorded. Verapamil (10(-5) to 10(-4) M) and adenosine (10(-5) to 10(-4) M) produced significant (P less than 0.05) shifts to the right of the histamine dose-response curve in normal physiological salt solution (PSS). Adenosine (10(-4) M) had no effect on the contractile responses to histamine in calcium-deplete PSS but significantly (P less than 0.01) increased the rate of relaxation (-dT/dt, 16.1 +/- 2.3 mg/s before adenosine, 53.7 +/- 7.0 mg/s during adenosine). In calcium-free PSS, verapamil (10(-4) M) had no effects on histamine-induced contractions, nor did it affect the spontaneous rate of relaxation. These findings suggest that the relaxant responses to adenosine, like verapamil, are partially mediated through blockade of external calcium influx, while adenosine, unlike verapamil, appears to have an additional intracellular mode of action.     

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.