Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo

Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.     

Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes

The effects of zinc on the rate of production of bactericidal O2- of polymorphonuclear leukocytes (PMN) in response to three different types of stimulating agents (serum-treated zymosan (STZ), Con A, and myristate) were studied. The percentage reduction of O2- production of PMN stimulated by STZ, Con A, and myristate were all reduced in response to Zn, irregardless of whether Zn was added to the reaction mixture immediately before SZT addition or following a prior 20 min. incubation of PMN in the presence of Zn. However, when Zn was introduced intraperitonially into guinea pigs before the collection of PMN from the animal, zinc treatment produced inhibition only in STZ-activated PMN; it produced no effect in O2- production of PMN stimulated by myristate, and it further augmented the O2- production stimulated by Con A.     

Plasma uric acid levels in ethanol-fed turkey poults treated with allopurinol

Plasma uric acid levels were determined in ethanol-fed poults following administration of allopurinol. In young poults, allopurinol at a dose of 50 mg/kg significantly depressed plasma uric acid levels 6 hr post-dosing. At 11 hr post-dosing, plasma uric acid levels were significantly elevated in the allopurinol-treated poults when compared with control poults. During a period of ethanol abstinence, allopurinol at a dose of 40 mg/kg significantly depressed plasma uric acid levels up to 8 hr post-dosing. At a dose of 30 mg/kg, plasma uric acid levels were similar to control values at 4 and 6 hr post-dosing. Data suggest that plasma uric acid levels can be depressed in ethanol poults when allopurinol is administered every 8 hr at a dose of 40-50 mg/kg of body weight.     

Partial monosomy 22 as result of an X/22 translocation in a newborn with DiGeorge syndrome

The case of a neonate with clinical symptoms of DiGeorge syndrome is reported. During pregnancy the measurements by ultrasonography revealed already a significant growth retardation of the fetus, for the first time obvious in the 20th week. The child died immediately after birth. A de novo translocation X/22 was observed with the translocation chromosome being late replicating in all mitoses analysed. The own observation is discussed regarding other cases with DiGeorge syndrome and taking the differential diagnoses into account. count.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.     

The "manganese(III)-containing" purple acid phosphatase from sweet potatoes is an iron enzyme

An improved purification of the purple acid phosphatase from sweet potatoes has been developed, and the properties of the enzyme have been reexamined. Contrary to previous reports, (e.g., Y. Sugiura, et al., J. Biol. Chem., 256, 10664-10670 (1981) ), the enzyme contains two moles of iron and insignificant amounts of manganese. The specific activity of the iron-containing preparations is ca. 14 times higher than that reported previously for the purported "Mn(III)" enzyme. The sweet potato purple acid phosphatase does indeed bind manganese, but it can be removed by dialysis with no changes in specific activity or spectral properties.     

Repeating structure of chick tropoelastin revealed by complementary DNA cloning

A cDNA library was constructed from chick aorta poly(adenylic acid)-containing RNA in the expression vector pEX1. Several clones were identified by screening the library with a polyclonal antiserum raised against chick tropoelastin and confirmed by DNA sequencing. Analysis of the deduced amino acid sequence, corresponding to the mature tropoelastin and most of the signal peptide, revealed that the molecule is composed of at least 8, and possibly 13, repeating units. The common features of each unit include an N-terminal region composed largely of alanines and lysines and ending with an aromatic amino acid, followed by a GAG span and then a C-terminal region consisting mostly of valines, prolines, and glycines often present in several copies of the sequence (VPGV). This structure is discussed in terms of the functional properties of the molecule.     

Sterol-phospholipid interaction in model membranes: role of C5-C6 double bond in cholesterol

Several double-bond isomers of cholesterol where the normal C5-C6 double bond (delta 5) has been moved to different positions in the ring skeleton, i.e., delta 1, delta 4, delta 7, delta 8(9), delta 8(14), and delta 14, have been synthesized and incorporated in phosphotidylcholine vesicles. In addition, dienes like delta 5,7, delta 7,14, and delta 8,14 have also been studied. Many of these cholesterol analogues are intermediates in the sterol biosynthesis in different organisms. The incorporation studied indicated that more than 90% of the sterol was present in the vesicles. The effect of these cholesterol analogues was studied by glucose permeability, electron spin resonance, and fluorescence polarization spectroscopy. These studies indicated that delta 14-cholesten-3 beta-ol was most effective in restricting glucose permeability or in increasing the order parameter but was still not as effective as cholesterol. This was followed by delta 8(14)- and delta 8(9)-cholesten-3 beta-ol. The delta 1, delta 4, and delta 7 analogues and the dienols were relatively less effective in condensing the membrane. These studies indicate that the double bond at C5-C6 in cholesterol is most effective for optimal sterol-phospholipid interaction and may have formed the basis of the migration of the double bond from rings C and D in sterols to C5-C6 during the evolution of cholesterol.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.