Torsion angle approach to nucleic acid distance geometry: TANDY

Summary An efficient algorithm for generating DNA structures from a given set of distance constraints has been developed. The present implementation is suited for single-stranded DNA. The performance of the program has been tested with constraint sets representative of most stringent theoretical cases and also with usually available experimental ones. The results indicate that use of NOE-derived constraints alone generates an extremely large family of conformers and suggest that the quality of structure determination may be enhanced by incorporating additional constraints obtained by other means. The speed of the program makes it ideal for interactive use in conjunction with other complementary algorithms such as those for spectral simulation, energy minimization and molecular dynamics calculations.Dedicated to the memory of Professor V.F. Bystrov     

The synthesis and biological activity of four novel fluorescent vasopressin analogs

We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.     

pH-dependent semiquinone formation by methylamine dehydrogenase from Paracoccus denitrificans. Evidence for intermolecular electron transfer between quinone cofactors

The quinonoid confactors of Paracoccus denitrificans methylamine dehydrogenase exhibited a pH-dependent redistribution of electrons from the 50% reduced plus 50% oxidized to the 100% semiquinone redox form. This phenomenon was only observed at pH values greater than 7.5. The semiquinone was not readily reduced by addition of methylamine, consistent with the view that this substrate donates two electrons at a time to each cofactor during catalysis. Once formed at pH 9.0, no change in redox state from 100% semiquinone was observed when the pH was shifted to 7.5, suggesting that the requirement of high pH was for formation and not stability of the semiquinone. The rate of semiquinone formation exhibited a first-order dependence on the concentration of methylamine dehydrogenase, indicating that this phenomenon was a bimolecular process involving intermolecular electron transfer between reduced and oxidized cofactors. The rate of semiquinone formation decreased with decreasing ionic strength, suggesting a role for hydrophobic interactions in facilitating electron transfer between methylamine dehydrogenase molecules. Methylamine dehydrogenase was covalently modified with norleucine methyl ester in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). This modification did not affect the catalytic activity of the enzyme but greatly inhibited the intermolecular redistribution of electrons at high pH. This modification also prevented subsequent cross-linking by EDC of the large subunit of methylamine dehydrogenase to amicyanin, the natural electron acceptor for this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.     

Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Infant development in the lion-tailed macaque,Macaca silenus (Linnaeus): The first eight weeks

Infant development in lion-tailed monkeys was observed during the first eight weeks after birth. A rapid decrease in the time spent in the closest association by the mother and the infant was seen between the first and second weeks. Between the second and fifth week there was a somewhat steady state with only marginal decrease in the time spent by the infant out of lap of the mother. However, during this time, a rapid decline in maternal cradling and restraining behaviour was seen. From the sixth week onwards, there was a rapid increase in the time spent by the infant out of contact with the mother. Independence of the infant during troop progression was seen only towards the eighth week, that too only marginal. Among the troop members the subadult females and the juvenile females showed the maximum interest in the infant. The former spent much more time than the juveniles interacting with the infant. The adult females showed only little interest in the infant while the adult male showed no interest at all. Infant snatching by subadult females and juvenile females was seen on many occasions. The infant remained in the possession of them from less than 1 min to more than 2 hr. This behaviour tended to decrease towards the sixth week.     

Isolation of fraction no. 10 from Taenia saginata and evaluation of its specificity for the diagnosis of bovine cysticercosis

In an attempt to prove the specificity of the crude Taenia saginata antigen for the immunodiagnosis of bovine cysticercosis, a major and highly immunogenic fraction (F10), responsible for the formation of the typical "long band" reaction in immunoelectrophoresis, has been isolated from T. saginata proglottides by immunoaffinity chromatography. The immunoabsorbent was prepared by coupling a specifically raised hyperimmune serum (HIS) anti-F10 to Sepharose 4B. The purity of the isolated F10 was demonstrated by immunoprecipitation reactions. The HIS anti-F10, however, cross-reacted with several larval and adult Taenia spp. Consequently, F10 showed cross-reactions with the sera of animals infected with hydatid cysts or larval T. hydatigena. F10 also reacted with HIS anti-F5 (Echinococcus granulosus) but was shown to be non-identical with the well known F5 of E. granulosus. These data prove that F10 of T. saginata was not species-specific but showed a group specificity for the taeniid family - a situation analogous to F5 of E. granulosus.     

Evolution of chromosomal abnormalities in sequential cytogenetic studies of ataxia telangiectasia

Summary Sequential cytogenetic studies of four patients with ataxia telangiectasia showed the progressive development of lymphocyte clones, each marked with a rearranged chromosome 14. Initial studies had shown random chromosomal breaks and rearrangements. Later studies in all patients showed nonrandom rearrangement of chromosome 14 with a breakpoint at 14q12 and with the distal segment translocated to either chromosome 14 or 7. The proportion of circulating lymphocytes carrying the marker tended to increase with time, accounting for the majority of the lymphocytes eventually in one case. The marked lymphocyte clones evolved further, as a result of loss of the small centric portions of the rearranged chromosome 14 (14pter14q12).Perhaps the abnormal clones in ataxia telangiectasia escape immunologic surveillance and flourish in an immunologically impaired environment. Subsequent to the loss of the centric portion of the rearranged chromosome 14, the cells may acquire additional capabilities that enhance malignant transformation.