Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

In mammalian skeletal muscle,neuronal-type nitric oxide synthase (nNOS) is found to be enriched atneuromuscular endplates. Here we demonstrate the colocalization of thenicotinic acetylcholine receptor (nAChR, stained with -bungarotoxin)and nNOS (stained with a specific antibody) in murineC₂C₁₂ myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C₂C₁₂myotubes. An antibody to the ₁-subunit of the nAChR didnot coprecipitate nNOS, and an nNOS-specific antibody did notprecipitate the ₁-subunit of the nAChR. Treatment ofmice with bacterial LPS downregulated the expression of nNOS inskeletal muscle, and treatment of C₂C₁₂ cellswith bacterial LPS and interferon- markedly decreased nNOS mRNA andprotein expression. In contrast, mRNA and protein of the nAChR (-,-, and -subunits) remained unchanged at the mRNA and proteinlevels. These data demonstrate that nNOS and the nAChR are colocalizedin murine skeletal muscle and C₂C₁₂ cells but differ in their expressional regulation.

     

Costs of immune response in cold-stressed laboratory mice selected for high and low basal metabolism rates

To study whether mounting an immune response is energetically costly, mice from two lines divergently selected for high (H-BMR) and low (L-BMR) basal metabolic rate (BMR) were immunized with sheep red blood cells. Their energy budgets were then additionally burdened by sudden transfer from an ambient temperature of 23 degrees C to 5 degrees C. We found that the immune response of H-BMR mice was lower than that of L-BMR mice. However, the interaction between line affiliation and ambient temperature was not significant and cold exposure did not result in immunosuppression in either line. At 23 degrees C the animals of both lines seemed to cover the costs of immune response by increasing food consumption and digestive efficiency. This was not observed at 5 degrees C, so these costs must have been covered at the expense of other components of the energy budget. Cold exposure itself elicited a considerable increase in food intake and the mass of internal organs, which were also heavier in H-BMR than in L-BMR mice. However, irrespective of the temperature or line affiliation, immunized mice had smaller intestines, while cold-exposed immunized mice had smaller hearts. Furthermore, the observed larger mass of the liver and kidneys in immunized mice of both lines kept at 23 degrees C was not observed at 5 degrees C. Hence, immunization compromised upregulation of the function of metabolically active internal organs, essential for meeting the energetic demands of cold. We conclude that the difficulties with a straightforward demonstration of the energetic costs of immune responses in these animals stem from the extreme flexibility of their energy budgets.     

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.     

Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.     

Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site

The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).     

Molecular characterization of binding of calcium and carbohydrates by an early activation antigen of lymphocytes CD69

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K(d) approximately 54 microM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Site-directed mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.     

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.     

Universality classes in folding times of proteins

Molecular dynamics simulations in simplified models allow one to study the scaling properties of folding times for many proteins together under a controlled setting. We consider three variants of the Go models with different contact potentials and demonstrate scaling described by power laws and no correlation with the relative contact order parameter. We demonstrate existence of at least three kinetic universality classes that are correlated with the types of structure: the alpha-, alpha-beta-, and beta- proteins have the scaling exponents of approximately 1.7, 2.5, and 3.2, respectively. The three classes merge into one when the contact range is truncated at a reasonable value. We elucidate the role of the potential associated with the chirality of a protein.