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PubMed1996

Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons.

Intermediate filaments comprise an integral part of the neuronal cytoskeleton. However, little is known about their function, and there remains some uncertainty about their precise subcellular localization. We examined the timing of expression and distribution of alpha-internexin, neurofilament triplet proteins and peripherin using immunocytochemistry in cultured hippocampal neurons. alpha-Internexin immunostaining was present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites which extended into dendritic spines. The presence of alpha-internexin in dendritic spines was confirmed in situ by electron microscopy of rat hippocampal tissue sections and suggests that this intermediate filament may serve as a link between cytoskeletal elements in dendritic shafts and spines. In culture, immunostaining using antibodies against individual triplet protein subunits indicated that light (NF-L) and middle (NF-M) subunits were first expressed in cells shortly after the initiation of axonal outgrowth. Expression of the heavy (NF-H) subunit occurred a few days later. Although timing and localization of expression did not correlate with the initiation of axonal or dendritic processes, it was coincident with periods of rapid outgrowth. Triplet proteins were more abundant in axons and appeared to be incorporated into lengthier filaments than in dendrites. Highly phosphorylated NFH/M immunoreactivity was polarized to axons after 6 days in culture. The distribution of one NF-H epitope was restricted to GABAergic neurons in mature cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results show that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of alpha-internexin suggests that it may be involved in the formation or maintenance of dendritic spines.

Animals↗

PubMed1975

Triplet state quenching by photoinduced radicals in the sensitized ethanol photolysis.

UV irradiation of a rigid solution of 2-amino-pyrimidine in ethanol at 77 degrees K results in a sensitized production of ethyl radicals. The radicals are formed via a biphotonic process. The increase of the radical yield with irradiation time is directly correlated to a decrease of the number of triplet state molecules as detected by ESR. A kinetic model of the biphotonic process is presented and verified by experimental results. It supports the hypothesis of a catalytically enhanced radiationless decay process of the triplet states due to an interaction between the radicals and the triplet state molecules.

Energy Transfer↗

PubMed2000

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimentional structures of proteins and its implications for secondary structure prediction from amino acid sequences.

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected non-homologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for a helix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For b-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.

Amino Acid Sequence↗

PubMed1999

On negative selection against ATG triplets near start codons in eukaryotic and prokaryotic genomes.

The frequencies of ATG triplets in the genomes of various species were systematically analyzed, and the frequency of ATG triplets was significantly low around start codons in both prokaryotic and eukaryotic genomes. In eukaryotes, however, the frequency decrease before the start codon is much more evident than that after the start codon. In prokaryotes, on the other hand, the ATG frequency pattern around the start codon is less evident, and-more importantly-symmetric. We also computed average distances between a start codon and its nearest upstream-located ATG triplet and found a general tendency for the average distances to be longer in higher organisms.

Animals↗

PubMed2005

Evolution of the genetic triplet code via two types of doublet codons.

Explaining the apparent non-random codon distribution and the nature and number of amino acids in the 'standard' genetic code remains a challenge, despite the various hypotheses so far proposed. In this paper we propose a simple new hypothesis for code evolution involving a progression from singlet to doublet to triplet codons with a reading mechanism that moves three bases each step. We suggest that triplet codons gradually evolved from two types of ambiguous doublet codons, those in which the first two bases of each three-base window were read ('prefix' codons) and those in which the last two bases of each window were read ('suffix' codons). This hypothesis explains multiple features of the genetic code such as the origin of the pattern of four-fold degenerate and two-fold degenerate triplet codons, the origin of its error minimising properties, and why there are only 20 amino acids.

Amino Acids↗

PubMed2006

The GAA triplet-repeat is unstable in the context of the human FXN locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model.

Friedreich ataxia (FRDA) is caused by homozygosity for FXN alleles containing an expanded GAA triplet-repeat (GAA-TR) sequence. Patients have progressive neurodegeneration of the dorsal root ganglia (DRG) and in later stages the cerebellum may be involved. The expanded GAA-TR sequence is unstable in somatic cells in vivo, and although the mechanism of instability remains unknown, we hypothesized that age-dependent and tissue-specific somatic instability may be a determinant of the progressive pathology involving DRG and cerebellum. We show that transgenic mice containing the expanded GAA-TR sequence (190 or 82 triplets) in the context of the human FXN locus show tissue-specific and age-dependent somatic instability that is compatible with this hypothesis. Small pool PCR analysis, which allows quantitative analysis of repeat instability by assaying individual transgenes in vivo, showed age-dependent expansions specifically in the cerebellum and DRG. The (GAA)(190) allele showed some instability by 2 months, progressed at about 0.3-0.4 triplets per week, resulting in a significant number of expansions by 12 months. Repeat length was found to determine the age of onset of somatic instability, and the rate and magnitude of mutation. Given the low level of cerebellar instability seen by others in multiple transgenic mice with expanded CAG/CTG repeats, our data indicate that somatic instability of the GAA-TR sequence is likely mediated by unique tissue-specific factors. This mouse model will serve as a useful tool to delineate the mechanism(s) of disease-specific somatic instability in FRDA.

Age Factors↗

PubMed2002

Triplet repeats and bipolar disorder.

Anticipation, the phenomenon of a disease becoming more severe or having earlier onset as it is transmitted down the generations, was originally described in families with psychiatric illness but was thought due to ascertainment bias and became forgotten. Interest was rekindled when a number of neurodegenerative disorders that show this phenomenon, were found to be due to a novel form of mutation--unstable triplet repeats showing intergenerational expansion. Some recent studies of anticipation are consistent with its occurrence in bipolar disorder but are still associated with methodological problems making interpretation difficult. A number of case-control studies employing the repeat expansion detection (RED) technique have found longer repeats in bipolar probands but other studies have found no such association. Despite a large number of studies examining the role of various repeat containing candidate genes, a pathogenic triplet repeat has yet to be found for bipolar disorder. It is likely that the controversy surrounding anticipation and the existence of triplet repeats will only finally be resolved with the demonstration of such a mutation in the aetiology of bipolar disorder.

Bipolar Disorder↗

PubMed1976

Kinetics of populating and depopulating of the components of the photoinduced triplet state of the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas spheroides (wild type), and its mutant R-26 as measured by ESR in zero-field.

Optically detected ESR spectra in zero magnetic field of the triplet state of three photosynthetic bacteria are presented. The zero field splitting parameters [D] and [E] and the widths of the resonances show small but significant differences for the three bacteria. The resonance lines are inhomogeneously broadened as demonstrated by hole-burning experiments. The populating probabilities and depopulating rates for the triplet sublevels have been measured. The populating kinetics are very similar for the three bacteria. The depopulating rates are more than one order faster than those of chlorophyll a and chlorophyll b and of porphyrin model systems. The populating probability of the lowest level is about 6 times less, and the depopulating rate about 6 times slower, than for the upper levels, identifying this level as the level connected to the molecular z-axis perpendicular to the plane of the molecule. The relative populations of the triplet sublevels are almost equal in zero magnetic field.

Electron Spin Resonance Spectroscopy↗

PubMed1981

Optically excited triplet states in the bacteria Rhodopseudomonas sphaeroides 'wild-type' detected by magnetic resonance in zero-field.

Optically-detected magnetic resonance (ODMR) experiments in zero-field on the photosynthetic bacteria Rhodopseudomonas sphaeroides revealed triplet states belonging to molecules which have a prompt emission in the optical region from 590 to 700 nm and a delayed emission between 700 and 830 nm. The zero-field parameters of these triplet states are 29 less than [D] less than 34 . 10(-3) cm-1 and 4 less than [E] less than 8 . 10(-3) cm-1, the decay rates of the [D] + [E] transitions being in the order of 60-340 ms. The correlation between optical emission and radio-frequency was used to separate the total optical emission from 590-700 nm into individual emissions, belonging to molecules whose triplet states were studied by ODMR in this region. Comparing the fluorescence microwave double resonance (FMDR) spectra with the results of excitation spectroscopy, as well as comparing the zero-field parameters and the decay rates with that of Mg-porphyrins in matrices given in the literature, allowed the identification of the emitting molecules as Mg-porphyrins which are produced by the biosynthesis of bacteriochlorophyll in the cells.

Magnetics↗

PubMed1982

Induction of neurofilament triplet proteins in PC12 cells by nerve growth factor.

The localization of neurofilament triplet proteins in PC12 cells grown in the absence of (PC12-) or maintained in the presence of (PC12+) nerve growth factor (NGF) was studied using indirect immunofluorescence and monospecific, immunosorbent purified antibodies to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins. The intensity of immunofluorescent staining of the triplet proteins was always greater in PC12+ compared with PC12-cells. Neuritic staining was seen in PC12+ cells with all 3 monospecific antibodies to neurofilament proteins. However, the perikaryal distribution of each of the neurofilament proteins differed in both PC12+ and PC12-cells. Monospecific antibodies to P68 protein yielded a 'ball-like' cytoplasmic staining pattern whereas monospecific antibodies by P150 protein stained in a stippled pattern. Monospecific antibodies to P200 on the other hand diffusely stained the perikaryal cytoplasm with very faint but detectable foci of 'ball-like' configurations and stippling. Electron microscopic study of PC12+ and PC12-cells revealed intermediate filaments in the cell bodies of both as well as in the processes of the former. 'Ball-like' clusters of such filaments were rarely seen. However, these filaments lacked the three-dimensional organization typical of intact neurofilaments. It is concluded that PC12 cells contain dissociated or incompletely assembled immunoreactive neurofilament triplet proteins and that these proteins can be induced by NGF. The PC12 cells are therefore an attractive model system not only for studies of neuronal differentiation but also for studies of neurofilament metabolism and disorders thereof.

Adrenal Gland Neoplasms↗

PubMed1993

Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'-CGG-3')17 oligodeoxyribonucleotide.

The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy (Kennedy's disease) and Huntington's disease. The molecular mechanisms underlying these triplet amplifications are still unknown. We demonstrate here that a synthetic (CGG)17 oligodeoxyribonucleotide can be utilized as hybridization probe to visualize some of the triplet repeats in the human genome. This technique may help in studies aimed at the elucidation of the amplification mechanism.

Autoradiography↗

PubMed1988

Induction of the SOS function sfiA in E. coli by systems which generate triplet ketones.

Generation of triplet ketones, either chemically through thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane and 3-[N-(pyridino)carbamoyl]methyl-3,4,4-trimethyl-1,2-dioxetane++ + or enzymatically via the aerobic oxidation of isobutyraldehyde trimethylsilyl enol ether catalyzed by horse-radish peroxidase, triggers the SOS function sfiA in E. coli. Although the observed effects are relatively weak and the triplet ketone scavenger tryptophan was ineffective in this system, our results provide evidence for the involvement of triplet ketones in this type of DNA damage. Possible mechanisms are discussed.

Antioxidants↗

PubMed1982

Probing the microenvironment of benzo[a]pyrene diol epoxide-DNA adducts by triplet excited state quenching methods.

Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25 +/- 1 degrees C were investigated: covalent adducts derived from the reaction of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BaPDE) and of (+/-)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of KTO2 approximately equal to 10(9) M-1 X S-1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of KTO2 is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of KTO2 approximately equal to 10(8) M-1 X S-1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The KTO2 values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1 X 10(-4)-3 X 10(-3) M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of KTO2 for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics.

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC+, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1-4) have been extended to triple helical polynucleotides.

Base Sequence↗

PubMed1979

The pH dependence of the reactions of flavin triplet states with amino acids. A laser flash photolysis study.

The pH dependence of the rate constants of reaction of several amino acids with the triplet states of flavin mononucleotide in aqueous solution has been determined. In addition, the relative contributions of hydrogen atom transfer, electron transfer and physical deactivation to the overall process of triplet quenching by amino acids have been estimated. Analogous experiments to those with amino acids were carried out with EDTA as the substrate. The results indicate that the flavin triplet state abstracts an electron from EDTA but does not form an excited state flavin-EDTA complex as suggested in a previous study.

Amino Acids↗

PubMed1986

Interaction between enzyme-generated triplet carbonyls and molecules intercalated into DNA.

The phosphorescence from enzyme-generated and -protected triplet acetone is very efficiently quenched by dyes intercalated into DNA. The process is unlikely to be due to energy transfer and is tentatively ascribed to electron transfer occurring within the DNA helix complex with the acting enzyme. This quenching markedly protects DNA from breaks induced by triplet acetone. In the case of some barely emissive enzyme-generated triplet carbonyl species, it is possible to detect a weak emission resulting from the interaction with dye X DNA; this emission may be associated with back electron transfer.

Acetone↗

PubMed1995

Nonrandom patterns of simple and cryptic triplet repeats in coding and noncoding sequences.

Triplet repeats of the sequence purine, purine, and pyrimidine [RRY(i)] are frequent and often polymorphic in humans. Some RRY(i) are composed predominantly of a continuous repeat of one sequence [simple RRY(i)], but the majority are cryptic RRY(i) that are not obvious until the bases are classified into R or Y before the full extent of the repeat becomes apparent. RRY(i) can be divided into 18 classes based on predominant nucleotides. These classes are highly nonrandom in abundance and in location within genes. In humans, simple or cryptic RRY(i), in which AAT or AAC triplets predominate, are preferentially located 3' of Alu repeats. RRY(i) with a predominance of AGC or GGC show a dramatic enrichment in coding sequence, and GGC also shows a dramatic enrichment in 5' untranslated regions of genes. Characterization of RRY(i) present in coding regions identify 10 protein motifs (An, Dn, Hn, Pn, Qn, Tn, GnS0-3Gm, (G/S)n, (S/G/N)n, and (L/P)n). Six of the protein motifs appear predominantly in DNA-binding proteins/transcription factors. Alignment of homologous protein sequences from other mammals reveals that both simple and cryptic RRY(i) are a major source of deletions or insertions in the genes that contain them. Cryptic RRY(i) may be candidates for triplet repeat genetic diseases and, when mutated in somatic cells, may contribute to carcinogenesis.

Animals↗

PubMed1994

Detection of a reduced-flavin triplet state in free flavins and DNA photolyase.

A transient species was observed upon laser excitation (lambda exc = 355 nm) of reduced flavins (cFIH2) under anaerobic conditions. The transient decayed with a lifetime of about 1 microseconds, was quenched by iodide ions and was assigned as the reduced flavin triplet state 3cFIH2. The triplet-state absorption spectrum assumed three forms, depending on the pH of the solution, corresponding to 3cFIH2 and its monoprotonated and monodeprotonated forms. The triplet-state lifetime was not sensitive to the presence of pyrimidine cyclobutane dimers, apparently ruling out its involvement in the dimer splitting reaction observed with model systems of reduced flavins. An analogous species is apparently formed in the reduced form of Escherichia coli DNA photolyase.

Anaerobiosis↗