A nuclear magnetic resonance study of the glucose metabolism of Hymenolepis diminuta exposed to histamine and serotonin in vitro.

The direct effects of the inflammatory mediators, histamine (HI) and serotonin (SE), on the glucose metabolism of Hymenolepis diminuta in vitro were studied by analyzing the excretory products from culture media, containing D-1-13C-glucose and various concentrations of HI and/or SE, by 1H-nuclear magnetic resonance (n.m.r.) spectroscopy. The results revealed that HI markedly accelerated the glycolysis process by increasing the amount of lactate production. The increased glycolytic activity was reflected in a concentration-dependent increase in glucose uptake. Excretion of acetate was also stimulated by HI. A low concentration of SE significantly increased succinate, acetate and lactate excretions, whereas a high concentration had little effect on lactate production and significantly decreased succinate and acetate excretions. A combination of HI and SE treatment at a low concentration had no significant effect, but at a high concentration showed an additive effect, with an increase in lactate production, a decrease in succinate production and an increase in glucose uptake. Thus this work confirms that HI and SE directly influence, albeit differently, energy metabolism of the tapeworm H. diminuta.     

Nutrition and inflammatory events: highly unsaturated fatty acids (omega-3 vs omega-6) in surgical injury.

Given the poor prognosis and high cost of care for patients with acute inflammatory responses (often leading to organ failure and/or allograft rejection), immunomodulation of this hyperresponse represents an important priority for research in nutritional medicine. The primary goal of nutritional support in inflammatory disease is to provide adequate energy, particularly through use of novel lipids (to alter eicosanoid pathway toward a more regulated inflammatory state), and protein to meet endogenous requirements for tissue repair IL-1 production, and restored cellular function, thus preventing secondary infection (52). Manipulation of macrophage eicosanoid production by use of omega-3 PUFA may reduce the cellular immune response (by competing with arachidonic acid, which produces inflammatory eicosanoids of the 2- and 4-series), whereas inclusion of MCT found in coconut oil may lower the arachidonic acid content of membrane phospholipids. As more data are obtained on the use of such tailored therapies in critically ill patients, a new generation of parenteral and enteral diets will be developed to reduce inflammation and immune dysfunction.     

The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity

We have analyzed the de novo telomere synthesis catalyzed by the enzyme telomere terminal transferase (telomerase) from Tetrahymena. Oligonucleotides representing the G-rich strand of telomeric sequences from five different organisms specifically primed the addition of TTGGGG repeats in vitro, suggesting that primer recognition may involve a DNA structure unique to these oligonucleotides. The sequence at the 3' end of the oligonucleotide primer specified the first nucleotide added in the reaction. Furthermore, the telomerase was shown to be a ribonucleoprotein complex whose RNA and protein components were both essential for activity. After extensive purification of the enzyme by a series of five different chromatographic steps, a few small low abundance RNAs copurified with the activity.     

Dynamics of telomere length variation in Tetrahymena thermophila

We have analyzed the mechanism and dynamics of telomere length variation in the macronucleus of Tetrahymena thermophila. In a newly differentiated macronucleus, the average length of the telomeric repeated sequence, (C4A2 X T2G4)n, is closely regulated. In contrast, in vegetatively dividing cells in log phase, all macronuclear telomeric sequences lengthen coordinately by 3-10 bp per generation until up to 1000 bp are added. In both elongated and short telomeres, characteristic single-stranded breaks on both strands are distally located. Reduction of elongated telomeres to their original length involves either the appearance of a novel type of variant cell, incapable of net telomere elongation, or, under stationary phase conditions, a reversible removal of telomeric sequences. The demonstration that telomeres are dynamic structures provides evidence for a model of telomere length regulation by activities that add and remove telomeric repeats.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding

Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.     

An overhanging 3'' terminus is a conserved feature of telomeres.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).