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PubMed1969

23Na nuclear magnetic resonance relaxation studies of sodium ion interaction with soluble RNA.

Interactions between (23)Na(+) and soluble RNA in aqueous solution are studied with the use of (23)Na nuclear magnetic resonance. At low concentrations of NaCl, the interactions obey a simple equilibrium model with a formation constant log (K(f))(3) = 2.8 +/- 0.3. The relaxation rate of the bound sodium is found to be T(1B) (-1) = 222 +/- 19 sec(-1) compared to that of free sodium T(1F) (-1) = 17.5 sec(-1). At high NaCl concentrations, the system deviates from the model, possibly owing to aggregation of the soluble RNA.

Magnetic Resonance Spectroscopy↗
PubMed1989

[Rapid destroying of mRNAs in disaggregated Dictyostelium cells and effect of antibiotics].

Nogalamycin can inhibit RNA synthesis in both aggregated and disaggregated Dictyostelium discoideum cells. Cycloheximide and puromycin are effective in blocking translation. The late mRNAs, specific for the multicellular stage, degrade rapidly in disaggregated Dictyostelium cells. Nogalamycin or actinomycin D and daunomycin protect some of these mRNAs. Cycloheximide stabilizes all of the mRNAs, Puromycin, however, does not protect the mRNAs from destroying. These results imply that the rapid degradation of the late mRNAs during cell differentiating does not depend on nuclease or protein synthesis de novo.

Anti-Bacterial Agents↗
PubMed1997

Mode of cytostatic action of mesoionic oxatriazole nitric oxide donors in proliferating human hematopoietic cells.

The cytopathological effects of the novel nitric oxide (NO)-releasing, mesoionic 3-aryl-substituted oxatriazole-5-imine derivatives GEA 3162 and GEA 3175, and a reference NO donor SIN-1 were investigated in proliferating human hematopoietic cells. The GEA compounds (10-50 microM) induced rapid surface changes, which progressed as peculiar deep indentations and strictures in human leukemic T cells (MOLT-3) in 30 min. An excess of red cells partially prevented these surface changes. GEA 3162-treated MOLT-3 cells became permeable to ethidium bromide and lost their ability to be stained by acridine orange after 5 h of exposure. GEA 3162 and GEA 3175 suppressed thymidine and uridine incorporation in a dose-dependent manner, reflecting the inhibition of DNA and RNA synthesis respectively. In addition, the GEA compounds inhibited the growth of human bone marrow stem cells, CFU-GM colonies being more susceptible to the cytostatic action than BFU-E. The reference compound SIN-1 had comparative cytostatic effects at ten times greater concentrations (500 microM). We conclude that NO-releasing mesoionic oxatriazole derivatives have cytostatic action against human malignant and non-malignant hematopoietic cells, supporting the value of NO-releasing and NO-inducing compounds as anti-cancer agents.

Antineoplastic Agents↗
PubMed1978

Role of intracisternal A-particles and of RNA molecules in plasmacytoma-associated immunodeficiency.

A subcellular fraction from murine plasmacytoma cells was shown to suppress the primary antibody response when injected into normal mice. The active subcellular fraction copurified with intracisternal A-particles. The RNA extracted from subcellular fractions enriched in A-particles was also immunosuppressive. This activity was due to a population of RNA molecules that contained polyadenylic acid. Upon fractionation on a sucrose gradient, two populations of immunosuppressive RNA were obtained with sedimentation velocities of 12 to 18S and 40 to 50S. The 40 to 50S RNA was shown to be a thermolabile aggregate of molecules that contained the 12 to 18S RNA molecules. Plasmacytoma-derived material with similar physicochemical characteristics had previously been shown to induce in normal mouse lymphocytes surface immunoglobulins with the plasmacytoma idiotype, supporting the possibility that one of the mechanisms responsible for the development of immunological deficiency is the change of surface immunoglobulins of nonmalignant B-cells.

Animals↗
PubMed1994

T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand.

The induction of immunoglobulin E (IgE) switching in B cells requires at least two signals. The first is given by either of the soluble lymphokines interleukin 4 (IL-4) or IL-13, whereas the second is contact dependent. It has been widely reported that a second signal can be provided by the CD40 ligand (CD40L) expressed on the surface of T cells, mast cells, and basophils. A defect in the CD40L has been shown recently to be responsible for the lack of IgE, IgA, and IgG, characteristic of the childhood X-linked immunodeficiency, hyper IgM syndrome (HIGM1). IgE can however be detected in the serum of some HIGM1 patients. In this study, we isolated T cell clones and lines using phytohemagglutinin (PHA) and allergen, respectively, from the peripheral blood of one such patient who expressed a truncated form of CD40L, and investigated their ability to induce IgE switching in highly purified, normal tonsillar B cells in vitro. Unexpectedly, 4 of 12 PHA clones tested induced contact-dependent IgE synthesis in the presence of exogenous IL-4. These clones were also shown to strongly upregulated IL-4-induced germline epsilon RNA and formed dense aggregates with B cells. Of the four helper clones, three were CD8+, of which two were characteristic of the T helper cell 2 (Th2) subtype. Two allergen-specific HIGM1 T cell lines, both of the Th0 subtype, could also drive IgE synthesis when prestimulated using specific allergen. All clones and lines were negative for surface expression of CD40L, and the mutated form of CD40L was confirmed for a representative clone by RNase protection assay and sequencing. The IgE helper activity could not be attributed to membrane tumor necrosis factor alpha (TNF-alpha) although it was strongly expressed on activated clones, and the addition of neutralizing anti-TNF-alpha antibody did not abrogate IgE synthesis. These results therefore suggest the involvement of T cell surface molecules other than CD40L in the induction of IgE synthesis, and that these molecules may also be implicated in other aspects of T-B cell interactions.

Base Sequence↗
PubMed2000

NO-aspirin protects from T cell-mediated liver injury by inhibiting caspase-dependent processing of Th1-like cytokines.

BACKGROUND & AIMS: Concanavalin A (con A)-induced hepatitis is an immunomediated disease in which assembly of CD4(+) T cells and T helper (Th)1-like cytokines causes Fas-mediated liver cell death. Nitric oxide (NO) modulates Th1 response in vitro. NCX-4016 is an NO-aspirin derivative that spares the gastrointestinal tract and shares molecular targets with NO. The aim of this study was to investigate whether this NO-aspirin modulates Th1-like response induced by con A. METHODS: BALB/c mice were injected with 0.3 mg con A per mouse alone or in combination with NO-aspirin (18-100 mg/kg) or aspirin (10-55 mg/kg). RESULTS: NO-aspirin, but not aspirin, caused a dose-dependent protection against liver damage induced by con A. At a dose of 100 mg/kg, NO-aspirin caused a 40%-80% reduction of interleukin (IL)-1beta, IL-12, IL-18, interferon (IFN)-gamma, and tumor necrosis factor alpha production without affecting cytokine messenger RNA expression. NO-aspirin prevented Fas, Fas ligand, and IL-2 receptor up-regulation on spleen lymphocytes and Fas ligand on hepatocytes and caused the S-nitrosylation/inhibition of IL-1beta-converting enzyme-like cysteine proteases (caspases) involved in the processing and maturation of IL-1beta and IL-18. IL-18 immunoneutralization prevented IFN-gamma release and protected from liver injury induced by con A. In contrast to a selective caspase 1 inhibitor, zVAD.FMK, a pancaspase inhibitor, prevented IFN-gamma release and protected the liver from injury. CONCLUSIONS: Th1-like response induced by con A is mediated by IL-18 and requires activation of multiple caspases. NCX-4016 causes the S-nitrosylation/inhibition of caspases involved in cytokine production. Inhibition of Th1-like response is a new anti-inflammatory mechanism of action of NO-aspirin.

Amino Acid Chloromethyl Ketones↗
PubMed1988

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.

Base Sequence↗
PubMed1981

The interaction of RNA polymerase and DNA. Effects on the helix-coil transition and light scattering.

To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transition of poly[d(A-T] bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA. The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized. This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points. The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized. Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions. Core enzyme, on the other hand, aggregate extensively with double-stranded DNA, the only under conditions of low salt (10 mM KCl), without Mg2+, or at high salt (300 mM KCl), with or without Mg2+, can this aggregation be eliminated. Core enzyme also does not aggregate in the presence of single-stranded DNA. These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase.

Binding Sites↗
PubMed1982

Behavior of RNA and protein synthesis during the acclimatization of the carp. Studies with isolated hepatocytes.

1. Carp hepatocytes were isolated by dissociating the liver tissue with collagenase. The procedure yields viable cells with highly preserved ultrastructural and metabolic features. The isolated cells were able to self-aggregated and form tissue. 2. RNA and protein synthesis activity was significantly higher in the carp hepatocytes from summer acclimatized fish compared to the activity present in the cold adapted animals. 3. RNA synthesis assayed in carp hepatocytes suspensions obtained from summer and winter acclimatized fish exhibited a behaviour consistent with an inverse compensation to the cold acclimatization state, being apparently repressed, whereas protein synthesis did not show a compensatory activity.

Acclimatization↗
PubMed1997

Pheromone-inducible expression of an aggregation protein in Enterococcus faecalis requires interaction of a plasmid-encoded RNA with components of the ribosome.

Transfer of the conjugative plasmid pCF10 from Enterococcus faecalis donor strains is induced by a peptide pheromone (cCF10) secreted by recipient cells. High-efficiency transfer requires expression of an aggregation protein (Asc10) encoded by the prgB gene and positively regulated by genes in a region 3-5 kb upstream, containing prgQ-R-S. Transcriptional fusion data reported here support the results of recent molecular analysis of the 5' ends of prgB transcripts which indicated that prgB transcription occurs by readthrough from the prgQ promoter. A 530-nucleotide prgQ-encoded RNA molecule (Q(L)) with rRNA-like domains is required for Asc10 production. Q(L) and cCF10 were found to interact with the L6 and S5 ribosomal proteins, respectively. Mutational analysis of Q(L) indicates that this RNA may also directly interact with 16S RNA. Q(L) is present in ribosomes translating the prgB message, and pheromone cCF10 may affect the association of this RNA with translation complexes. Results suggest that the positive regulatory molecules act post-transcriptionally on the polycistronic message and modify a ribosome population to enhance pheromone-induced translation of prgB.

Amino Acid Sequence↗
PubMed2000

YC-1, a benzyl indazole derivative, stimulates vascular cGMP and inhibits neointima formation.

The pathobiologic process of arterial stenosis following balloon angioplasty continues to be an enigmatic problem in clinical settings. This research project investigates the ability of YC-1, a benzyl indazole derivative that sensitizes sGC/cGMP, to stimulate endogenous cGMP and attenuate balloon injury-induced neointima (NI) formation in the rat carotid artery. Northern and Western blot analyses revealed enhanced acute expression of iNOS and inducible heme oxygenase (HO-1) mRNA and protein in the injured artery. The contralateral uninjured artery also demonstrated acute HO-1 mRNA and protein induction without detectable iNOS expression. Perivascular application of YC-1 immediately following injury significantly stimulated acute vessel wall cGMP compared to untreated controls. YC-1 treated sections demonstrated significant reduction in NI area (-74%), NI area/medial wall area (-72%), and NI thickness (-76%) 2 weeks post-injury. These results directly implicate YC-1 as a potent new therapeutic agent capable of reducing post-angioplasty stenosis through endogenous CO- and/or NO-mediated, cGMP-dependent processes.

Angioplasty, Balloon↗
PubMed1993

Clustered arginine residues of bacteriophage lambda N protein are essential to antitermination of transcription, but their locale cannot compensate for boxB loop defects.

The N protein coded by bacteriophage lambda plays an essential role in the completion of lambda transcription by recognizing the boxB sequence in nascent transcripts and then aggregating with Escherichia coli RNA polymerase and four other E. coli proteins into an unstoppable transcription complex. In order to explore the functionality of N protein and the specific recognition between N and boxB, 14 amino acid positions near the amino-terminal end of N lambda were mutated extensively. The mutant proteins were scored for N function in vivo by a two-plasmid construct that visualizes readthrough transcription as lacZ expression in colonies of E. coli. Mutation was achieved by single TAG replacements, translated through suppression into 13 different amino acids, or by scrambling at assorted three-codon sets. Of the 14 amino acid positions tested (Tables 5 and 6), six remained functional with a wide variety of substitutions, while substitution was sometimes deleterious at one Ala and two Gln positions. At each of the five Arg positions, however, maintenance of Arg occupancy proved important for N function. Despite effective screening for increased N function at boxBs with defective loops, no N mutant, simple or complex, was found to change the order of preference of wild-type N lambda for boxBs with defective loops. Thus, although multiple amino-terminal Arg positions are found to be important for N function, mutations in the region spanning the five Arg residues were not found to compensate for defects in boxB loop.

Amino Acid Sequence↗
PubMed1994

Pituitary adenylate cyclase activating polypeptide (PACAP) potently enhances tyrosine hydroxylase (TH) expression in adrenal chromaffin cells.

In primary cultured bovine adrenal chromaffin cells (BACC), pituitary adenylate cyclase activating polypeptide 1-38 (PACAP) produced a dose related increase in tyrosine hydroxylase (TH) Vmax when measured 48 hours after the beginning of the treatment; a significant increase was observed with 0.5 nM and the maximal induction of close to 2.5-fold was found with 0.1 microM PACAP. The potency of PACAP was nearly 3 orders of magnitude greater than forskolin and VIP in inducing TH activity. These effects were preceded by an increase in TH mRNA levels, that started 2 hours after treatment and peaked 12 hours later. The presence of the phosphodiesterase inhibitor HL 725 further increased the stimulation of TH activity by PACAP, indicating that this activation was mediated via a cascade of events initiated by cAMP. Nicotine (1 microM) failed to increase TH activity significantly, however, when added in association with PACAP, a statistically significant increase of TH was elicited with peptide concentrations 5 times lower (0.1 nM) than the threshold dose of the peptide. The stimulation of nicotinic receptors facilitates the TH induction elicited by PACAP.

Adrenal Medulla↗
PubMed1990

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

Animals↗
PubMed2004

Matrix metalloproteinase 2 is suppressed by trapidil, a CD40-CD40 ligand pathway inhibitor, in human abdominal aortic aneurysm wall.

BACKGROUND: The activation of inflammatory cells and the production of matrix metalloproteinases (MMPs) are important in the pathogenesis of abdominal aortic aneurysm (AAA). Previous studies have demonstrated that the antiplatelet agent trapidil has multiple actions, including suppression of MMP expression through the inhibition of the CD40-CD40 ligand (CD40-CD40L) pathway in cultured cells. A recent clinical study suggested that trapidil might have functions beyond its antiplatelet action. Methods and results In the present study, we performed immunohistochemical analysis and semiquantitative reverse transcription-polymerase chain reaction to evaluate the effect of trapidil on the production of MMPs in cultured aortic tissues from patients with infrarenal AAA (n = 9) and control patients with aortoiliac occlusive disease (n = 7). The tissue concentrations of both MMP-2 and MMP-9 were significantly higher in AAA walls than in control aortic walls. Both trapidil and an anti-CD154 (CD40L) antibody significantly suppressed the protein production and mRNA expression of MMP-2 but did not inhibit those of MMP-9 in organ cultures of AAA wall specimens. MMP-9 was produced by macrophages and a lot of neutrophils in AAA tissues, whereas MMP-2 was derived from macrophages. CD40 was expressed on macrophages but not on neutrophils, and this expression could explain the differential effect of trapidil on the production of MMP-2 and MMP-9. CONCLUSIONS: Trapidil, a CD40-CD40L pathway inhibitor, suppressed mRNA expression and protein production of MMP-2 in AAA tissues, suggesting a potential therapeutic approach for the prevention or treatment of AAA.

Aortic Aneurysm, Abdominal↗
PubMed2004

Comparative analysis of the in vivo angiogenic properties of stable prostacyclin analogs: a possible role for peroxisome proliferator-activated receptors.

OBJECTIVE: Until recently, prostacyclin (PGI2) biological activities were thought to be exclusively mediated by cell surface receptors named IP. Recent studies have instead identified a novel pathway of PGI2 signaling, occurring through activation of peroxisome proliferator-activated receptors (PPARs) located in the nucleus. The availability of stable PGI2 analogs with different affinity for IP receptors and PPARs provides the possibility to test the importance and function of this dual pathway in vitro and in vivo. In this study, the in vivo angiogenic properties of different PGI2 analogs and the potential relationship between PPAR-mediated pathways, vascular endothelial growth factor (VEGF), and angiogenesis were investigated. METHODS AND RESULTS: By using the murine corneal model of angiogenesis, we found that PGI2 analogs able to act on nuclear PPARs, such as iloprost and carbaprostacyclin (cPGI), induce angiogenesis in vivo. In contrast, cicaprost, a PGI2 analog that only acts on IP receptors, has no in vivo angiogenic activity. Interestingly, angiogenesis induced by iloprost and cPGI does not differ in extent and morphology from that induced by VEGF and is associated with local increment of VEGF mRNA expression and protein levels. Finally, iloprost-induced angiogenesis is significantly decreased by systemic inhibition of VEGF activity, obtained by gene transfer of a soluble form of the VEGF receptor Flt-1. CONCLUSIONS: These data demonstrate that stable PGI2 analogs may have angiogenic properties in vivo, depending on their ability to act on PPARs. The resulting angiogenic process appears to be mediated by VEGF. These findings indicate that important physiological activities in the cardiovascular system, such as angiogenesis and VEGF induction, may be modulated by PGI2 through specific activation of the PPAR signaling pathway in vivo, with potentially important fundamental and clinical implications.

Animals↗
PubMed1999

Interleukin-12 is synthesized by mesangial cells and stimulates platelet-activating factor synthesis, cytoskeletal reorganization, and cell shape change.

Preliminary studies indicate the involvement of interleukin (IL)-12 in experimental renal pathology. In the present study, we evaluated whether cultured glomerular mesangial cells are able to produce IL-12 and whether IL-12 may regulate some of their functions, including the cytoskeletal reorganization, the change in cell shape, and the production of platelet-activating factor (PAF). The results obtained indicate that pro-inflammatory stimuli, such as tumor necrosis factor-alpha and bacterial polysaccharides, induce the expression of IL-12 mRNA and the synthesis of the protein by cultured mesangial cells. Moreover, cultured mesangial cells were shown to bind IL-12 and to express the human low-affinity IL-12 beta1-chain receptor. When challenged with IL-12, mesangial cells produced PAF in a dose- and time-dependent manner and superoxide anions. No production of tumor necrosis factor-alpha and IL-8 was observed. Moreover, we demonstrate that IL-12 induced a delayed and sustained shape change of mesangial cells that reached its maximum between 90 and 120 minutes of incubation. The changes in cell shape occurred concomitantly with cytoskeletal rearrangements and may be consistent with cell contraction. As IL-12-dependent shape change of mesangial cells was concomitant with the synthesis of PAF, which is known to promote mesangial cell contraction, we investigated the role of PAF using two chemically different PAF receptor antagonists. Both antagonists inhibited almost completely the cell shape change induced by IL-12, whereas they were ineffective on angiotensin-II-induced cell shape change. In conclusion, our results suggest that mesangial cells can either produce IL-12 or be stimulated by this cytokine to synthesize PAF and to undergo shape changes compatible with cell contraction.

Angiotensin II↗