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Triplet repeat disorders: discussion of molecular mechanisms.

Comparison of the growing number of disorders known to be associated with triplet repeat expansions reveals both common features and a diversity of molecular pathways. Despite significant progress towards the characterization of proteins coded by the mutant genes, the complex nature of these disorders requires identification of all molecular components of the triplet repeat pathways. In this brief review we will discuss recent progress in determining the molecular mechanisms of disorders with unstable trinucleotide mutations.

Animals↗

Measuring the coding potential of genomic sequences through a combination of triplet occurrence patterns and RNY preference.

The distribution of n-tuplet frequencies is shown to strongly correlate with functionality when examining a genomic sequence in a reading-frame specific manner. The approach described herein applies a coarse-graining procedure, which is able to reveal aspects of triplet usage that are related to protein coding, while at the same time remaining species independent, based on a simple summation of suitable triplet occurrences measures. These quantities are ratios of simple frequencies to suitable mononucleotide-frequency products promoting the incidence of the RNY motif, preferred in the most widely used codons. A significant distinction of coding and noncoding sequences is achieved.

Algorithms↗

A triplet chemotherapy with cisplatin, docetaxel and gemcitabine in patients with advanced non-small-cell lung cancer: a phase I/II study.

PURPOSE: We conducted a phase I/II study of triplet chemotherapy consisting of cisplatin (CDDP), docetaxel (DCT) and gemcitabine (GEM) in patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Fifty-three untreated patients with stage IIIB or IV NSCLC were enrolled. All drugs were given on days 1 and 8. The doses of CDDP and DCT were fixed at 40 mg/m(2) and 30 mg/m(2), respectively. In the phase I portion, a dose escalation study of GEM with starting dose of 400 mg/m(2) was conducted and primary objective in the phase II portion was response rate. RESULTS: The maximally tolerated dose (MTD) and recommended dose (RD) of GEM were determined as 800 mg/m(2) because grade 3 non-hematological toxicity (liver damage, diarrhea, and fatigue) developed in three of nine patients evaluated at that dose level. In pharmacokinetic analysis, C (max) and AUC of dFdC and dFdU were increased along with the dose escalation of GEM. However, no relationship between pharmacokinetic parameters and toxicity or response was observed. Objective response rate was 34% and median survival time was 11.7 months. Though major toxicity was myelosuppression, there were no life-threatening toxicities. CONCLUSION: These results indicate that this triplet chemotherapy is feasible and effective in patients with advanced NSCLC.

Adult↗

Small increase in triplet repeat length of cerebellum from patients with myotonic dystrophy.

Myotonic dystrophy (DM) is genetically characterized by abnormal expansion of an unstable CTG trinucleotide repeat, located in the 3'-untranslated region of mRNA encoding the family of serine-threonine protein kinases. DNA extracted from various organs of patients with DM was analyzed by the Southern blotting method. We identified differently expanded bands in DNAs from various tissues from patients with DM. In studying the length of the CTG repeat in different regions of the brain, we found a noticeably small increase in repeat length in the cerebellum compared with other tissues. While this phenomenon has been reported in other triplet repeat diseases such as Huntington disease, spinocerebellar ataxia type 1, and dentatorubral-pallidoluysian atrophy, we are the first to describe it in DM. Although the mechanism of expansion of the triplet repeat remains to be defined, the tissue-dependent somatic mosaicism suggests that its occurrence may depend on the differentiated state of each tissue.

Cerebellum↗

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines.

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)n and (CGG)n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome.

Base Sequence↗

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin.

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction 3Zncyt + az(II)-->Zncyt+ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrostatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.

Azurin↗

Triplet pregnancy involving complete hydatidiform mole and two fetuses: genetic analysis by deoxyribonucleic acid fingerprint.

A case of a triplet pregnancy involving a dizygous twin pregnancy and a complete hydatidiform mole after therapy with human menopausal gonadotropin and human chorionic gonadotropin is reported. Two female fetuses, two placentas in one mass with two amnions and two chorions, and a tumor mass with a grapelike appearance were spontaneously delivered at 19 weeks of gestation. The deoxyribonucleic acid fingerprints of the two placentas and tumor tissue were compared with those of the parents. The fingerprints of the placentas showed patterns different from each other; however, all their polymorphic fragments could be traced back to either the father or mother. All polymorphic fragments of the tumor tissue were inherited only from the father (androgenesis). These results indicated that this triplet pregnancy involved a dizygous twin pregnancy and a complete hydatidiform mole.

Adult↗

Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration.

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 M urea and a linearly increasing sodium phosphate concentration from 10 to 400 mM at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 M urea, and completely separated with 4 M urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 M urea at first, and then P 160 and P 68 were separated in the presence of 4 M urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 M.

Amino Acids↗

Measurement and analysis of triplet-state lifetimes by multifrequency cross-correlation phase and modulation phosphorimetry.

In this paper we describe a novel approach to study the triplet-state lifetimes by a conventional multifrequency cross-correlation phase and modulation apparatus. The analysis of phase and modulation data of eosin-labeled band 3 erythrocyte ghosts revealed the existence of two phosphorescence lifetime values of 2700 and 750 microseconds, with a fractional contribution of 78 and 22%, respectively, which are in good agreement with those reported in the literature. Differential polarization phase analysis, which facilitates the study of the rotational properties of band 3, provided data in good agreement with those reported in the literature. The method proposed in this paper to study the radiative emission from the triplet state may represent a convenient alternative to the pulse laser flash technique.

Erythrocyte Membrane↗

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase.

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.

1-Propanol↗

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2 degrees K.

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2 degrees K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are D = 0.02794 +/- 0.00007 cm-1, E = 0.00382 +/- 0.00007 cm-1, i.e. equal to those of monomeric chlorophyll a to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperatures 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.

Chloroplasts↗

On the magnetic field dependence of the yield of the triplet state in reaction centers of photosynthetic bacteria.

The yield of the triplet state in reaction centers of Rhodopseudomonas sphaeroides is dependent on the strength of an applied magnetic field. Reaction centers of the wild type that lack a functional iron complexed to the primary acceptor ubiquinone show a dependence similar to that of reaction centers of the mutant R-26 in which the iron-ubiquinone complex is intact. Apparently, the iron of the iron-ubiquinone complex is not essential to the effect, but it does exert an influence on its extent. Inchromatophores, the effect is about 2-fold decreased; the value of the magnetic field at which half the effect is found is about 500 G, in contrast to this value for reaction centers, which is 50--100 G. The magnetodependence of the triplet yield is discussed in terms of the Chemically Induced Dynamic Electron Polarization mechanism (CIDEP).

Bacteriochlorophylls↗

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I. II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters.

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77-84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with [D] = 279 X 10(-4) cm-1 and [E] = 39 X 10(-4) cm-1. The triplet resonances titrate with a midpoint potential of +380 +/- 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and delta Hpp = 1.0 mT. Deconvolution of the P-700+ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species A0, which is characterized by g = 2.0033 +/- 0.0004 and delta Hpp = 1.0 +/- 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A1 in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A1 is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A1 in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A1 from its binding site.

Chlorophyll↗

Photodegradation of DNA with fluorescent light in the presence of riboflavin, and photoprotection by flavin triplet-state quenchers.

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, alpha-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.

Butylated Hydroxyanisole↗

Partial purification and characterization of an ovarian tripeptidyl peptidase: a lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets.

The pregnant hog ovary is a rich source of a novel exopeptidase that catalyzes the release, at pH 5.0, of collagen-related (P3-P2-P1) "triplets" such as Gly-Pro-Met, Gly-Pro-Arg, and Gly-Pro-Ala from arylamide derivatives, provided the N termini are unsubstituted. Corresponding derivatives of related (P2-P1) dipeptides (Pro-Met, Pro-Ala) or (P1) amino acids (Met, Arg, Ala) are not attacked. The enzyme was purified 58-fold from a detergent extract of a water-extracted tissue residue. Activity was determined on Gly-Pro-Met-2-naphthylamide at pH 4.5 and 37 degrees C (Km 0.45 mM; Vmax 722 nmoles/min/mg protein). The responsible Mr 55,000 exopeptidase, termed "tripeptidyl peptidase", forms high-Mr aggregates, belongs to the serine catalytic class, and has a lysosomal localization. Gly-Pro-Ala triplets were released sequentially at pH 5.0 from a Mr 14,000 polypeptide, poly(Gly-Pro-Ala-). When this reaction was coupled to that of homologous dipeptidyl peptidase II, the liberated tripeptides were reduced to dipeptides and free amino acids: (Gly-Pro-Ala)n----nGly-Pro-Ala----nGly-Pro + nAla.

Aminopeptidases↗

Presence in invertebrate genomes of sequences characterized by the repetition of the triplet CCPurine.

In Drosophila melanogaster (Dm), polypeptidic domains have been found in different morphogenetic genes. Two types of them are characterized by the repetition of nucleotidic triplets: the M repeat (CAX) and the paired repeat (CAXCCX). In this paper we described a third type of repeat isolated from the genome of a Polychaete annelid: Owenia fusiformis. This repeat is characterized by the repetition of the triplet CCPurine. Phylogenetic studies showed the presence of this repeat in all the invertebrate genomes tested (eight copies in Dm genome) while we failed to detect it in vertebrate genomes.

Amino Acid Sequence↗

Assessment of immunological properties of neurofilament triplet proteins.

The relationship between mammalian neurofilament triplet proteins was studied immunologically using rabbit and guinea pig antibodies to bovine neurofilament triplet proteins. Neurofilament proteins were separated by preparative electrophoresis, each protein being isolated and re-electrophoresed to enhance purification. Antisera to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins showed greatest activity with the corresponding protein immunogen but also revealed cross-reactivity with the other two neurofilament proteins when assessed by the ELISA method. The same antigenic inoculum elicited variable cross-reactivity, more in the guinea pig than in the rabbit. Rabbit antisera to P68 was specific in that it did not cross-react with P150 or P200. Rabbit antisera to P150 and to P200 were rendered specific by absorption with P200 and P150, respectively. By electron microscopy, isolated neurofilaments became decorated with an uniform coat of antibodies when exposed to specific antisera for each of the neurofilament proteins. By indirect immunofluorescence, each antisera showed identical patterns of tissue localization, corresponding to the distribution of neurofilaments in peripheral nerve, spinal ganglia, spinal cord, cerebellum and cerebrum. Neurofilament antigens were not detected in liver, kidney, spleen, lung, bladder, intestine, aorta, heart or tongue.

Animals↗

Carbocyanine dyes used as fluorescent triplet probes for measuring slow rotational diffusion of lipids in membranes.

A method for measuring the slow rotational diffusion of lipids or lipid domains in membranes has been developed. It covers the time range from 20 microseconds-5 ms, and has a greater than 5 x 10(4) molecules of probe. The method uses acyl-substituted carbocyanine dyes as fluorescent triplet probes and a laser-microscope combination for excitation and measurement of the triplet state. Rotation rates in dimyristoylphosphatidylcholine vesicles were sensitive to the liquid-to-gel transition. Slow rotations with relaxation times of about 100 microseconds were detected at the transition temperature region.

Carbocyanines↗