Application of X-rays to dental age estimation in medico-legal practice.

AIM OF THE STUDY: The paper addresses the use of dental age assessment methods based on radiographs in medico-legal practice. Different cases of practical application of the methods are presented including identification of human remains, dental age assessment in a living person and one archaeological case. MATERIAL AND METHODS: The study material consisted of cases involving dental age assessment performed in the Department of Forensic Medicine, Poznan University of Medical Sciences in Poznan. Depending on the preliminary assessment of age, the Liversidge or the Kvaal et al. methods were applied. Dental age was estimated on the basis of available pantomograms. In the case of the living person, it was a radiograph supplied for expert evaluation. In the other cases, dental computed tomography was performed. RESULTS: Dental age was successfully estimated in all of the cases. Various methods based on the analysis of X-ray images were applied. Dental age was shown to be correlated with skeletal age. CONCLUSIONS: The methods based on radiographs were demonstrated to be useful, and the results they yield are fully correlated with results of anthropological analyses.

Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes.

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA-RNA ribozyme targeted to the HIV-1 5' LTR was investigated, using THP-1, THP-1/HIV-1IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1IIIB cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 microM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1IIIB cells to 5 microM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 microM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 microM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4+ cells immediately after infection with HIV-1IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.

Estimate of lifetime dose in persons exposed occupationally to x rays in Poland.

Estimates are presented of the magnitude of doses incurred over prolonged periods by individuals exposed occupationally to x rays. The results of individual monitoring in Poland from 1966 to 1978 formed the basis of the estimates. Probability distributions of receiving a certain 40-y lifetime x-ray dose were assigned to several subgroups monitored by applying the method of Markov's chains. Independently, by linear extrapolation of the measured increment rate, the mean life doses were estimated for a number of subgroups. The results seem to demonstrate that occupational exposure to x rays in the subgroups studied is slightly diversified, and its current level is of the same order as the exposure due to background radiation.

[Estimated number of expected stochastic changes in the population occupationally exposed to X-rays]. / Ocena liczby spodziewanych zmian stochastycznych w populacji osób zawodowo narazonych na dzialanie promieniowania rtg.

In this paper the life-time effective dose equivalent and expected postradiation stochastic effects number in various subpopulations occupationally exposed to X-rays have been estimated. The prognosis of life-time doses absorbed by those occupationally exposed to X-rays and the relationship between the effective dose equivalent and surface exposure dose registered in the individual dosimetry system constituted the basis for this evaluation. The obtained results indicate that under the present X-ray exposure conditions the probability of the occurrence of X-ray adverse health effects in form of post-radiation cancers is insignificantly low.

Polystyrene reverse-phase ion-pair chromatography of chimeric ribozymes.

The use of a reverse-phase polystyrene resin for ion-pair HPLC purification of large amounts of synthetic chimeric DNA-RNA oligomers that is faster and more reliable than previously used techniques has been developed. The preparation of synthetic oligomers containing RNA requires the use of tetrabutylammonium fluoride in the final step, the cleavage of the tert-butyl-dimethyl silyl protecting group from the ribonucleotides. Cleavage is accompanied by the serendipitous formation of ion pairs between tetrabutylammonium cations and the oligomer phosphates. The formation of these ion pairs retards the elution of the oligomer during HPLC, which allows rapid removal of excess tetrabutylammonium fluoride and the concomitant purification of chimeric ribozymes. This technique is based on a correlation between the length of ion-paired oligomers and their retardation during HPLC. The advantages of reverse-phase ion-pair HPLC on polystyrene resin for the fast purification of oligoribonucleotides are discussed and illustrated through the examples of synthesized chimeric ribozymes.

Studies directed toward introduction of N6-isopentenyladenosine and its 2-methylthio analogue into oligoribonucleotides.

A new efficient route of i6A and ms2i6A synthesis, especially useful for the preparation of i6A-containing oligoribonucleotides eg. Api6A has been proposed. The method is based on aminolysis of a 6-methylsulfonylpurine system formed by oxidation of its 6-methylthio precursor.

Incorporation of nonbase residues into synthetic oligonucleotides and their use in the PCR.

Oligonucleotides containing the nonbase residues 1,3-propanediol or 1,4-anhydro-2-deoxy-D-ribitol were synthesized and used as primers for the polymerase chain reaction (PCR). Since these residues cannot be replicated by a DNA polymerase, the resulting PCR products have protruding 5' ends. Primers were designed with three regions, a 3' region complementary to the desired template, a 5' region complementary to a preselected nucleotide sequence, and a nonreplicable element interposed between these two containing 1-3 of the nonbase residues. The primers were used in a PCR and the products hybridized without denaturation to a solid support containing an immobilized preselected nucleotide sequence. Studies are reported showing the effects of the nonreplicable elements in primer extension reactions and the application to the capture of PCR products.

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Correlation of activity with stability of chemically modified ribozymes in nuclei suspension.

To examine hammerhead ribozyme activity in the nuclear environment, we have used nuclei isolated from HTLV-I tax transformed fibroblasts to evaluate ribozymes targeted against HTLV-I tax RNA. The ribozyme activity in nuclei suspension was strongly dependent on the resistance of the particular ribozyme to endogenous nucleases. A ribozyme containing exclusively 2'-deoxynucleotides in its stems cleaved target RNA by its catalytic activity in the absence of proteins and caused degradation in their presence by induction of nuclear RNase H activity. A ribozyme containing 2'-amino- and 2'-fluoropyrimidine nucleosides in combination with terminal phosphorothioate linkages was significantly more stable in nuclei suspension and also exhibited a more than threefold higher cleavage efficacy than its unmodified counterpart. The increased resistance against nuclease degradation is mainly due to terminal phosphorothioate linkages, suggesting that both 5' and 3'-exonucleases are primarily responsible for the nuclear degradation of oligonucleotides.

Inhibition of transcription factor binding by ultraviolet-induced pyrimidine dimers.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for the induction of mutations and the development of skin cancer. Cis-syn cyclobutane pyrimidine dimers (pyrimidine dimers) are the most frequent lesions produced in DNA by UV irradiation. Besides being mutagenic, pyrimidine dimers may interfere with other important DNA-dependent processes. To analyze the effects of pyrimidine dimers on the ability of DNA sequences to be recognized by trans-acting factors, we have incorporated site-specific T-T dimers into oligonucleotides containing the recognition sequences of the sequence-specific transcription factors E2F, NF-Y, AP-1, NF kappa B, and p53. In each case, presence of the photodimer strongly inhibited binding of the respective transcription factor complex. Reduction of binding varied between 11- and 60-fold. The results indicate that the most common UV-induced DNA lesion can interfere severely with binding of several important cell cycle regulatory and DNA damage responsive transcription factors. We suggest that inhibition of transcription factor binding may be a major biological effect of UV radiation since promoter regions are known to be repaired inefficiently and since UV damage can deregulate the function of a large number of different factors.

Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway.

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line.

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

Ribozyme-mediated RNA degradation in nuclei suspension.

Ribozymes containing 2'-fluoro- and 2'-amino-modified pyrimidine nucleosides in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In order to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with proteinase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.