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PubMed1989

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters.

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.

Acridines↗
PubMed1990

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

Avian Sarcoma Viruses↗
PubMed1991

[Clinical usefulness of the measurement of plasma D-dimer levels].

To evaluate the clinical usefulness of D-dimer, various effects on the measurement of D-dimer were examined. Although both fibrinolytic and fibrinogenolytic products were detected by the measurement of FDP, only fibrinolytic products were detected by the measurement of D-dimer. In patients with DIC and other thrombo-embolic diseases, plasma D-dimer levels were significantly higher than in normal persons. A significant positive correlation between plasma D-dimer and serum FDP was found in DIC patients. In patients with DIC associated with acute promyelocytic leukemia, which is thought to be an increased fibrinogenolysis state, serum FDP was higher than the plasma D-dimer which suggests that increased fibrinogenolysis affects the result of serum FDP measurement. Plasma D-dimer significantly increased 5 minutes after endoscopic embolization with thrombin in the patients with esophageal varices. However serum FDP increased 30 minutes after the treatment, which suggests that the D-dimer is more useful for rapid detection of coagulo-fibrinolytic change than serum FDP. Plasma D-dimer was significantly higher in patients with cerebral infarction and increased with age. These finding suggest the usefulness of plasma D-dimer measurement for the specific and rapid evaluation of coagulo-fibrinolytic activation and thrombo-embolic state.

Adult↗
PubMed1990

[Measurement of FDP-D-dimer in DIC and pre-DIC].

We measured FDP-D-dimer value in disseminated intravascular coagulation (DIC), pre-DIC (within 7 days before onset of DIC) and suspected DIC (not completely satisfying the DIC criteria). The level of FDP in many patients with pre-DIC was normal, but the level of FDP-D-dimer in most patients with pre-DIC was increased. FDP was markedly increased one day before onset of DIC but FDP-D-dimer was increased 7 days before the onset. FDP was significantly higher in DIC than in pre-DIC, but it was not higher in pre-DIC than in suspected DIC. In patients with hematological malignancies, FDP-D-dimer was statistically higher in DIC than in pre-DIC and in pre-DIC than in suspected DIC, but in non-hematological malignancies, FDP-D-dimer was not significantly different among the 3 groups. The peak increase of FDP-D-dimer was noted at a DIC score of 7 or 8. The correlation of FDP-D-dimer with FDP was better in pre-DIC than in DIC, and the ratio of FDP-D-dimer to FDP was higher in pre-DIC. FDP-D-dimer was not correlated with fibrinopeptide A or B beta 15-42 in pre-DIC. It is speculated that pre-DIC is a hypercoagulable state and FDP-D-dimer may be useful to the diagnosis of pre-DIC.

Disseminated Intravascular Coagulation↗
PubMed1986

The repair of pyrimidine dimers via a DNA-glycosylase mechanism.

The "UV endonuclease" isolated either from M. luteus or bacteriophage T4 infected E. coli (the denV gene product) consists of two enzymatic activities on a single polypeptide chain: a pyrimidine dimer-DNA glycosylase and an AP endonuclease. The repair of pyrimidine dimers by this enzyme is initiated by the cleavage of the N-glycosylic bond of the 5' pyrimidine of the dimer that leaves the cyclobutane dimer still attached to the DNA through the N-glycosylic bond of the 3' pyrimidine of the dimer. This reaction results in the formation of an apyrimidinic site in the DNA. The second step in this repair pathway is the endonucleolytic cleavage of the DNA 3' to the AP site by the associated AP endonuclease. As a result, the nicked DNA contains DNA damage on both sides of the incision site: an apyrimidinic moiety on the 3' end and a thymine-thymidylate dimer on the 5' end. The enzymes prefer double stranded DNA over single stranded DNA, and thymine over cytosine at the 5' position of the dimer. The AP endonuclease activity prefers the AP site created by the pyrimidine dimer-DNA glycosylase on UV irradiated DNA over either apurinic or apyrimidinic DNA. This repair mechanism appears to be operative in vivo since DNA intermediates containing thymine-thymidylate dimer sites have been detected in UV irradiated T4 infected E. coli and in UV irradiated M. luteus. The cloned denV gene partially complements the UV repair deficient uvr A, B, C strains of E. coli.

Cloning, Molecular↗
PubMed1986

Excision of pyrimidine dimers from the DNA of Neurospora.

Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmunoassay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers. UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency.

DNA Repair↗
PubMed1978

The influence of inhibitors on dimer removal and repair of single-strand breaks in normal and bromodeoxyuridine substituted DNA of HeLa cells.

The elimination of cyclobutane pyrimidine dimers from the nuclear DNA of ultraviolet irradiated HeLa cells has been examined by means of chromatography and immunoautoradiography. The extent and duration of the process was similar when dimers were assayed by both methods, proving that the anti-sera recognized pyrimidine dimers. The rate of dimer excision did not differ through the cell cycle with the exception of mitosis during which no dimers were removed. Dimer excision is a relatively fast process which is terminated within a few hours, but it leaves many dimers in the DNA. Excision is depressed by inhibitors of semiconservative DNA synthesis that affect the DNA precursor pool or DNA polymerases. Cells whose DNA is partly substituted with bromodeoxyuridine instead of thymidine, repair single-strand breaks and remove dimers at the same rate but to different extents. On the other hand, inhibitors limit repair of breaks and removal of dimers to the same degree suggesting that the repair of the two types of lesion is coordinated.

Bromodeoxyuridine↗
PubMed1990

Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts.

UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, e.g., (6-4) photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

Cells, Cultured↗
PubMed1995

A study of the dimerization of Rous sarcoma virus RNA in vitro and in vivo.

The Rous sarcoma virus dimer linkage site (DLS) has been located by electron microscopy at position 511 +/- 28 nucleotides. We have studied the dimerization of RNAs encompassing the first 634 nucleotides of Rous sarcoma virus and conclude that there are at least two dimerization signals. One is located between nucleotides 531 and 634 and may involve Watson-Crick pairing of an imperfect inverted repeat. The other signal is located between nucleotides 496 and 530. A tetraguanine sequence at nucleotides 523-526 is required for dimerization of this domain. The guanines are not involved in an identifiable Watson-Crick interaction or in guanine tetrad formation. Either dimerization domain can initiate the dimerization of RNA 1-634. It is possible that these domains are two parts of a single dimerization signal. Interstrand RNA contacts within the virion are not limited to the DLS but occur along the length of the genome. Nascent virions contain monomeric RNA which slowly associates to form an RNA dimer. The limiting step in dimerization is not proteolytic cleavage of the gag precursor because only the mature capsid protein p27 can be detected in these nascent virions.

Avian Sarcoma Viruses↗
PubMed1975

Formation of dimers in ultraviolet-irradiated DNA.

Evidence has been obtained that in UV-irradiated native DNA, pyrimidine dimers are preferentially formed in long pyrimidine tracts. This effects is not the result of the existence of more dimerizable thymine in the long pyrimidine tracts. The preferential formation of dimers in long pyrimidine tracts is enhanced as the dose of irradiation is decreased. These results suggest that the formation of dimers in native DNA occurs by a cooperative mechanism. Quite likely, dimers are formed only in regions of native DNA that are locally denatured (breathing), in which the bases can be aligned. The formation of a dimer in such a breathing region would tend to lock it open and could lead to cooperative formation of dimers in such regions. If this mechanism for the cooperative formation of dimers is operative then it should be restricted to double-strand DNA. The pyrimidine tracts of irradiated single-stranded DNA all show virtually the same dimer content, irrespective of the length of the tract. These results are consistent with the hypothesis that dimers form cooperatively in breathing regions.

Binding Sites↗
PubMed1985

The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form.

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.

Animals↗
PubMed1987

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase.

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.

Animals↗
PubMed1990

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Base Sequence↗
PubMed1992

Rapid quantitative evaluation of plasma D-dimer levels in thrombotic states using an automated latex photometric immunoassay.

To evaluate a recently developed latex photometric immunoassay (LPIA) that which can measure 40 samples quantitatively within 30 minutes, we measured D-dimer levels in blood samples obtained from patients with disseminated intravascular coagulation (DIC). Linearity of D-dimer determination was shown over the range from 0.5 to 36 micrograms/ml, and recovery studies demonstrated 94 to 108% recovery. The intra-assay and inter-assay coefficients of variation ranged from 0.6 to 11.3% at plasma D-dimer levels of 0.54 to 30.1 micrograms/ml. No interference by lipids, bilirubin, haemoglobin, rheumatoid factor, or gamma-globulin was noted. The normal D-dimer range was less than 0.5 microgram/ml in healthy ambulatory subjects, while the level in elderly subjects with atherosclerosis (14%) or in immobilized subjects (38%) was well above this limit. There was a strong correlation between plasma and serum D-dimer levels (r = 0.993). D-dimer levels measured by this LPIA showed a good correlation with those determined using two kinds of ELISA. The LPIA D-dimer levels were elevated in some subjects with diseases predisposing to DIC, but remained below 10.0 micrograms/ml. On the other hand, the LPIA D-dimer levels in DIC subjects were almost always above 10.0 microgramS/ml. Our study showed that a hypercoagulable state should be suspected when the LPIA D-dimer level is greater than 0.5 microgram/ml and that DIC should be diagnosed when the level is greater than 10.0 micrograms/ml in the presence of an appropriate underlying disease. This LPIA system can rapidly evaluate the presence of a hypercoagulable state as accurately as any ELISA, and thus seems potentially valuable for both emergency and routine laboratory use.

Adult↗
PubMed1986

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide.

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.

Amino Acid Sequence↗
PubMed1968

Pyrimidine dimers in the DNA of Paramecium aurelia.

The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and "loss of photoreactivability of dimers." It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia.

Animals↗
PubMed1982

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I.

The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied. Only a dimeric form of the enzyme can be phosphorylated [Misset, O., Brouwer, M., & Robillard, G. T. (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J. S., & Robillard, G. T. (1981) Biochemistry 20, 87--93]. Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active. Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer. Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity. The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides. The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization.

Escherichia coli↗
PubMed1986

Dimeric form of diphtheria toxin: purification and characterization.

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.

Animals↗