Differential binding of mannose-specific lectins to the carbohydrate chains of fibrinogen domains D and E

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.     

Copepods of the Juréia ecological reserve,state of São Paulo,Brazil. II. The genera Hesperocyclops,Muscocyclops, and Bryocyclops (Cyclopoida,Cyclopidae)

Cyclopid copepods collected mainly in aquatic microcosms and semiterrestrial habitats in the Juréia Ecological Reserve are studied. Hesperocyclops herbsti and Bryocyclops campaneri are described as new species and their taxonomical relationships discussed. Females of Muscocyclops operculatus (Chappuis) are redescribed and the males described for the first time. An emended diagnosis for Muscocyclops is proposed.     

A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina

Dipolyunsaturated phosphatidylcholines from bovine retina contain a whole series of unusual fatty acids. Methyl esters from these acids are very strongly retained on polar and nonpolar gas-liquid chromatography stationary phases. On thin layers of silica-AgNO3, they separate as tetra-, penta-, and hexaenoic fatty acid methyl esters. After hydrogenation, the three polyunsaturated fractions give the same series of saturated methyl esters, having 20 (or 22)-36 carbon atoms. High pressure liquid chromatography, as well as gas-liquid chromatography, indicates that the new components of the three fractions are even-carbon homologs of well known polyenoic fatty acids of the n-6 and n-3 families, since they behave as series of 20-36-carbon tetraenoic (n-6), pentaenoic (n-3 and n-6), and hexaenoic (n-3) fatty acids. Their occurrence in phospholipid molecules also having docosahexaenoate (22:6) explains the separation of major dipolyunsaturated phosphatidylcholines from retina into dodecaenoic, undecaenoic, and decaenoic fractions after argentation thin layer chromatography. Using high pressure liquid chromatography, the latter are resolved into individual species having 10-12 double bonds and 42-58 carbon atoms. The unusual PCs are thus endowed not only with the highest degree of unsaturation, but with the longest hydrocarbon chains yet reported for vertebrate glycerophospholipids. It is shown that phosphatidylcholines containing the novel fatty acids are highly concentrated in photoreceptor membranes and that they occur in the retina of vertebrates so distant in evolution as fish, birds, and various mammals.     

Studies on evolutionary and selective properties of hypercycles using a Monte Carlo method

Summary The most relevant properties of hypercycles were previously studied mainly from a theoretical point of view. We have developed a Monte Carlo method simulating hypercyclic organization to obtain information about the dynamics of this prebiotic organization. Nucleation, growth, and selective properties have been tested and the results obtained are in good agreement with those of the theoretical predictions. The influence of hypercyclic organization of the error threshold has also been studied. As a consequence of the emergence of a hypercycle, the value of this threshold decreases. The amount of this decrease depends on the population size. Moreover, for some interval of quality factor values, either the hypercycle organization or an error catastrophe can be produced, depending on the initial conditions. The influence of these phenomena on both the dynamic behavior and evolutionary advantages of the hypercycle, as well as their decisive roles on genome size, are discussed.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic study of pancreatic proteinase loci,Prt-1, Prt-2, Prt-3, and Prt-6

Various patterns of mouse pancreatic proteinase activity bands were observed on agarose gel electrophoresis. Prt-1 ^a and Prt-1 ^b genes control the positive (PRT-1A) and negative (PRT-1B) expression of tryptic band V, respectively; Prt-2 ^a and Prt-2 ^b correspond to chymotryptic bands II (PRT-2A) and III (PRT-2B); Prt-3 ^a and Prt-3 ^b control the low (PRT-3A) and high (PRT-3B) tryptic activities of band IV; the Prt-1 and Prt-3 loci are closely linked on the same chromosome; Prt-6 ^a and Prt-6 ^b correspond to tryptic bands I (PRT-6A) and I (PRT-6B). Twenty-four laboratory strains from the United States showed the phenotype PRT-1A, PRT-3A, and PRT-2A. Of laboratory strains established in Europe, 6 showed PRT-1A, PRT-3A, and PRT-2A, and 10 had PRT-1B, PRT-3A, and PRT-2A bands. Most wild mice around the world and their descendants showed the phenotype PRT-1B, PRT-3B, and PRT-2A. Only the phenotype of M. m. brevirostris was PRT-1A, PRT-3A, and PRT-2A, which was the same as most laboratory inbred strains. PRT-2B was observed mainly in Japanese (M. m. molossinus) and Korean (M. m. yamashinai) wild mice. PRT-6B was detected only in Mus spicilegus and Mus caroli, but all other mice including wild populations and laboratory strains showed PRT-6A. New biochemical phenotypes such as PRT-2C and PRT-3C were also found in this study.     

Labeling of lipids of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n-6)) docosapentaenoate (22:5(n-3)) and docosahexaenoate (22:6(n-3))

(1-14C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20:4, 22:5 and 22:6, respectively) are efficiently taken up and actively esterified into the lipids of bovine retina after 2 h incubation. Photoreceptor membranes, mitochondria, microsomes and postmicrosomal supernatants, which display significant differences in phospholipid and fatty acid compositions, are isolated after such incubations to study the labeling of lipids. The lipid classes preferentially labeled with the acids (1) largely differ among and within subcellular fractions, while (2) some common features in the treatment of the three polyenes are observed in each fraction. In all of them, the three acids are actively incorporated in phosphatidylcholine; ethanolamine glycerophospholipid, phosphatidylserine (PS) and phosphatidylinositol (PI) are highly labeled with 22:6, 22:5 and 20:4 respectively; within ethanolamine glycerophospholipid, the three label phosphatidylethanolamine in preference to plasmenylethanolamine. Most of the 14C esterified in mitochondria is in phospholipids. The endoplasmic reticulum produces in addition highly labeled triacylglycerols, also found in cytosol. High levels of 14C-labeled diacylglycerols are observed exclusively in photoreceptor membranes, where the specific radioactivity of PI is very high. The total amounts of 14C incorporated (1) are in general similar within a given fraction for the three polyenes, but (2) largely differ among fractions. The labeling of the highly unsaturated phospholipids of photoreceptor membranes is the lowest, while the postmicrosomal supernatant (whose lipids are relatively the poorest in polyenoic fatty acids) contains most of the labeled lipids isolated from retinas under these conditions. The results indicate that polyunsaturated species of retina phospholipids undergo an active synthesis and turnover, as well as an intense intracellular traffic among membranes.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.