Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.     

Adenylate cyclase activity in cyanobacteria: activation by Ca(2+)-calmodulin and a calmodulin-like activity

An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.     

Structural analysis of plant ribosomal 5S RNAs. Visualisation of novel tertiary interactions by cleavage of lupin and wheat 5SrRNAs with ribonuclease H

A model for the tertiary structure of plant 5S rRNA, previously proposed by our laboratory (Joachimiak, A. et al. (1990) Int. J. Biol. Macromol., in press) was tested by specific cleavage of the plant 5S rRNA in the presence of synthetic oligodeoxynucleotides. The hexanucleotides used in this study were complementary to different portions of loops C, D and E, the nucleotides of which have recently been proposed to be involved in tertiary hydrogen bonds. The results obtained strongly support the interaction of loops C and D by nucleotides C34, C35, C36, A37 and G85, G86, G87, U88, respectively. Digestion pattern of loop E (domain gamma, nucleotides 66-110) suggests a possible different arrangement of this part of the plant 5S rRNA molecule, when compared with other eukaryotes.     

Studies on the N-acetyl-beta-D-hexosaminidase B from germinating Lupinus luteus L. seeds. II. Mechanism and inhibition with some 2-acetamido-2-deoxyaldono(1----4)lactones

The N-acetyl-beta-D-hexosaminidase B of germinating yellow lupin seeds catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and -galactosaminide substrates. The investigation of the pH dependence of the kinetic parameters (Vmax and Vmax/Km) demonstrated that two common ionizable groups (probably two carboxyl groups) play an essential role in the catalysis. That is, the enzyme has a lysozyme-like splitting mechanism, and the possibility of an anchimeric assistance provided by the acetamido group seems to be negligible. The presence of a deprotonated carboxyl group near the glycosidic linkage was also supported by inhibition with 1-thio substrate analogues. On the other hand, some 2-acetamido-2-deoxyaldono(1----4)lactones proved to be effective inhibitors of the hexosaminidase with the exception of the D-arabinose derivative, which can be explained by high stereospecificity in the binding.     

An immunochemical study of antigen expression in potato spindle tuber viroid (PSTV) - infected tomato leaves and calluses

A polyspecific antiserum against protein extracted from PSTV-infected tomato leaves was prepared and the IgGs were separated by affinity chromatography on a beaded cellulose adsorbent with an immobilized “healthy” antigen. The antibody not adsorbed entered into a preferential reaction with the antigen from PSTV-infected leaves as estimated by an enzyme-linked immunosorbent assay. The immunochemical reactions did not significantly exceed the control background, if antigens from tomato leaves infected with potato viruses X, Y and M were analyzed. By immunoblot technique we revealed, however, that several antigens not detected in healthy leaves appeared in the leaves infected either with PSTV or with viruses X and M. An accumulation of a major antigen having a molecular mass of about 70 kDa was observed in viroid-infected leaves only, suggesting the specificity for viroid infection. The antigen was found not to be an alkaline endoproteinase - the pathogenesis-related protein P-69. Some antigens with molecular masses approximately 38.0, 23.7 and 22 kDa, which occurred in PSTV-infected leaves and in healthy calluses, were not detectable in PSTV-infected calluses. No reaction exceeding the control level was observed using enzyme-linked immunosorbent assay for antigens from silver nitrate-treated tomato leaves, although such leaves showed symptoms similar to that caused by viroids.     

Helix-coil stability constants for the naturally occurring amino acids in water. XXIII. Proline parameters from random poly (hydroxybutylglutamine-co-L-proline)

Water-soluble random copolymers containing L-proline and N5-(4-hydroxybutyl)-L-glutamine were synthesized by copolymerization of the tripeptides H-L-Glu(OBzl)-L-Glu(OBzl)-L-Glu(OBzl)-OH and H-L-Glu(OBzl)-L-Pro-L-Glu(OBzl)-OH, using benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate as condensing reagent, and subsequent aminolysis of the Bzl ester groups with 4-amino-1-butanol. These copolymers were found to contain significant amounts of N5-(4-hydroxybutyl)-D-glutamine, thus requiring the synthesis of a binary copolymer containing only D- and L-N5-(4-hydroxybutyl)glutamine residues in order to evaluate the possible effects of the D-residues on the conformational properties of poly(hydroxybutylglutamine-co-L-proline). The different copolymers were fractionated, and their thermally induced helix-coil transition curves were obtained in water at neutral pH. When proper corrections were applied for the helix-destabilizing properties of N5-(4-hydroxybutyl)-D-glutamine, the Zimm-Bragg parameters sigma and s for L-proline could be deduced from the melting curves of poly(hydroxybutylglutamine-co-L-proline). The results indicate that L-proline acts as a very strong helix breaker over the entire temperature range from 0 to 60 degrees C.     

Interaction of surfactants with model and biological membranes. II. Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine bilayers as studied by spin probe ESR

The interaction of N-alkyl-N,N,N-trimethylammonium (CnTMA, n = 6-18) salts (iodides and/or bromides) with model membranes prepared by hydration of egg yolk phosphatidylcholine (EYPC) over aqueous salt solutions has been studied by m-doxyl stearic acid (m-DSA, m = 12 and 16) spin probe method. In disoriented EYPC bilayers the CnTMA salts decrease the orientational order parameter S33 of m-DSA evaluated from the powder pattern ESR spectra. This effect is maximal for C6TMA. In oriented EYPC bilayers prepared by the parallel-beam sputtering method and hydrated over saturated NaCl solution the order parameter S33 calculated from the angular dependence of the nitrogen hyperfine splitting is decreased in the presence of C6TMA. The order parameter S11 obtained from the angular dependence of line positions indicates deviation of m-DSA motion from axial symmetry. C6TMA increases the probability of gauche conformations of the lipid chains by about 13-14%, and decreases the effective energy difference between the trans and gauche conformations by about 420-480 J/mol, at molar ratio of EYPC/C6TMA = 2:1. The angular dependence of linewidths is analysed by employing a theory of spin relaxation based on the strong collision model for molecular reorientations. The correlation time tau 0 of the reorientation of an axis orthogonal to the doxyl ring of 16-DSA is decreased in the presence of C6TMA, while that of 12-DSA is not influenced by it. The ratio of tau 2/tau 0 is increased in the presence of C6TMA for the both spin probes. The results are explained using the free-volume model of the CnTMA-EYPC membrane interaction.     

Differences in the renal handling of pancreatic and salivary amylase in the rat.

Plasma activity and excretion of pancreatic (P) amylase in the rat was found to be negligible. In contrast, the excretion rate of salivary (S) amylase was substantial and variable, depending on diuresis. P-amylase had a higher isoelectric point, a greater sieving coefficient, and a shorter half-life than S-amylase. A bolus injection of 125I-labelled enzymes was followed by the appearance of 125I-labelled enzyme- as well as protein-free 125I activity in the urine. The enzyme loss was smaller and the fraction of protein-free 125I activity higher following injection of P-amylase. The affinity of P-amylase to paraffin oil exceeded that of S-amylase in partition experiments with water and paraffin oil in vitro. It is concluded that both renal filtration and reabsorption of P-amylase exceed those of S-amylase. This might be due to the higher lipophility of P-amylase in comparison to the salivary type.