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G Schlüter

Publications and source records attributed to G Schlüter.

At least 19 recordsLinked to original sources
PubMed2002

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Journal Article↗
PubMed2001

A 74-bp promoter of the Tnp2 gene confers testis- and spermatid-specific expression in transgenic mice.

During the final stages of spermatogenesis, round spermatids undergo several morphological, biochemical, and physiological modifications which result in the formation of mature spermatozoa. One of these is the nuclear condensation, achieved by the replacement of somatic-type and testis-specific histones by transition proteins and protamines leading to cessation of transcription several days before the completion of spermiogenesis. Therefore, a strict temporal and stage-specific gene expression is necessary for the correct differentiation of round spermatids into mature spermatozoa. In this study, the 5' regulatory region of rat Tnp2 gene was investigated by primer extension analysis and transgenic mice study. Primer extension analysis revealed a transcription start site which lies 70 bp upstream of the translation start codon. By transgenic mice studies, we demonstrated that a 147-bp 5' untranslated region corresponding to the region -74 to +73 is sufficient to confer testis- and spermatid-specific expression of rat Tnp2 gene.

3' Untranslated Regions↗
PubMed2001

Rat transition nuclear protein 2 regulatory region directs haploid expression of reporter gene in male germ cells of transgenic mice.

During spermiogenesis, the successive replacement of somatic histones by basic proteins, the transition nuclear proteins and protamines, allows normal sperm condensation. Transition nuclear protein 2 (TNP2) is transcribed postmeiotically in round spermatids. In order to determine regulatory flanking sequences responsible for stage specific expression of TNP2 gene, different transgenic mice were generated by microinjection of fertilized eggs. We demonstrate here that 525 bp of 5'- and 920 bp of 3'-flanking sequences of rat TNP2 gene could properly and efficiently direct chloramphenicol acetyltransferase gene expression to the postmeiotic male germ cells of transgenic mice. During male germ cell differentiation the first transgene transcripts were observed in round spermatids and translation started 6 days later in elongating spermatids, which is an evidence for posttranscriptional regulation of transgene expression. In contrast, transgenic mice bearing only the 525 bp 5'-flanking sequences of rat transition protein 2 gene and 3'-flanking sequences of the simian virus 40 large T antigen showed low levels of transgene expression in testis. From these results, it can be concluded that the 525 bp 5'-flanking sequences regulate the cell specific expression and the sequences located in 920 bp 3'-flanking region either enhance the transgene expression in the male germ cells or may have a posttranscriptional role in stabilizing the mRNA in addition to its function in delaying the mRNA translation. Using comparative alignment of 5'-flanking of TNP2 genes from different species, the putative regulatory sequences are identified.

3' Untranslated Regions↗
PubMed2001

Bioassay of mannitol and caprolactam and assessment of response to diethylnitrosamine in heterozygous p53-deficient (+/-) and wild type (+/+) mice.

Alternative bioassays of mannitol (MAN) and caprolactam (CAP) were conducted in transgenic p53-deficient mice. Also, to assess the sensitivity of the transgenic mice to a model DNA-reactive carcinogen, the hepatic effects of diethylnitrosamine (DEN) were compared in the wild type background strain of mouse and in the transgenic derivative. Fifty-one male wild type strain C57BL/6 mice p53 (+/+), 8 weeks old, and 51 heterozygous p53 (+/-) C57BL/6 Tac-[KO] Trp53 N5 mice, 8 weeks old, were allocated to six experimental groups as follows: groups 1 (wild type +/+) and 2 (p53 +/-) served as room controls, groups 3 (+/+) and 4 (+/-) were exposed orally (gavage) to 50 mumol/kg body weight DEN weekly for a total of ten doses during the first 10 weeks of the study, group 5 (+/-) was exposed to 15,000 ppm CAP in the diet for up to 26 weeks, and group 6 (+/-) was exposed to 50,000 ppm MAN in the diet for up to 26 weeks. After 10 weeks, liver from control and DEN-exposed mice was used for O4-ethylthymidine (O4-EtT) DNA adduct analysis by the immunoslot blot method. The cell replicating fraction (RF) in the liver was determined by quantification of the percentage of immunohistochemically stained hepatocytes positive for proliferating cell nuclear antigen. No significant or consistent body or liver weight changes were present in any of the treatment groups. No consistent and pertinent changes in RF values were present in any of the treatment groups. None of the tested substances produced neoplasms of any type in p53 (+/-) mice. DEN induced comparable levels of O4-EtT adducts in the liver in both wild type and p53 +/- genotypes, but no morphologic changes were evident in the livers of either genotype. The lack of response to DEN, in spite of formation of DNA adducts, may reflect the resistance to hepatocarcinogenesis of the background C57BL/6 strain of the transgenic, and calls into question the general sensitivity of this transgenic for detection of carcinogenic effects.

Administration, Oral↗
PubMed2000

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame.

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.

5' Untranslated Regions↗
PubMed2000

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin.

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.

Animals↗
PubMed2000

Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity.

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

Adenosine Triphosphate↗
PubMed1999

DNA analysis of Huntington's disease: five years of experience in Germany, Austria, and Switzerland.

OBJECTIVE: To review the direct DNA testing for Huntington's disease (HD) in Germany, Switzerland, and Austria from 1993 to 1997, and to analyze the population with regard to age structure, gender, and family history. METHODS: Twelve laboratories (nine in Germany, two in Austria, and one in Switzerland) recorded data pertaining to repeat number, gender, age at molecular diagnosis, and family history of probands. The molecular test was categorized as either diagnostic (for symptomatic individuals), presymptomatic (for individuals at risk), and prenatal (for pregnancies at risk). RESULTS: A total of 3,090 HD patients, 992 individuals at risk, and 24 fetuses were investigated using DNA analysis. The clinical diagnosis was confirmed in 65.6% of patients. A total of 38.5% of individuals at risk inherited an expanded CAG repeat. The female-to-male ratio showed a distinct predominance of women both in the diagnostic and presymptomatic groups. Of the fetuses tested, six were carriers of an expanded CAG repeat. Two pregnancies were interrupted; one pregnancy was not. No information about the parents' decision was obtained for the remaining three pregnancies. CONCLUSIONS: Approximately 20% of the estimated 10,000 HD patients living in Germany, Switzerland, and Austria have been identified by DNA analysis (total population, approximately 100 million; incidence of HD, 1:10,000). Assuming a ratio of HD patients to individuals at risk of 1:3, approximately 30,000 individuals are, in principle, eligible for a presymptomatic test. Less than 3 to 4% of individuals at risk have requested a presymptomatic test. This shows that the assumed enormous request of predictive testing has not occurred. More surprisingly, prenatal diagnoses were found to be rare.

Adult↗
PubMed1999

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver.

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. Löser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.

Animals↗
PubMed1999

Different mechanisms for the photoinduced production of oxidative DNA damage by fluoroquinolones differing in photostability.

Several fluoroquinolone antibacterial agents exhibit an adverse phototoxic effect in humans and are photo-cocarcinogenic in mice. The UV-induced production of reactive oxygen species plays a role in the toxicity and may be involved in carcinogenicity. Four fluoroquinolones were examined for the ability to photochemically produce oxidative damage in naked DNA. The major structural difference in the fluoroquinolones that would have an effect on their photostability is the functionality at the 8-position. At this position, 1-cyclopropyl-7-(2,8-diazbicyclo[4.3.0]non-8-yl)-6, 8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY y3118) contains a chlorine atom, lomefloxacin a fluorine atom, ciprofloxacin a proton, and moxifloxacin a methoxy group. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA was assessed by HPLC with electrochemical detection, and strand breaks were measured in pBR322 with agarose gel electrophoresis. The relative photolability of the fluoroquinolones correlated to the extent of production of 8-oxodGuo and strand breaks, with both UVA and UVB irradiation, in the following order: BAY y3118 approximately lomefloxacin > ciprofloxacin > moxifloxacin. Experiments were performed to determine whether the mechanism of damage was due to a type I (radical) or type II (singlet oxygen) pathway. Nitrogen depletion of oxygen resulted in a decrease in the extent of formation of 8-oxodGuo, suggesting that oxygen was involved. The use of selective radical or singlet oxygen inhibitors was inconclusive with respect to which pathway was involved. The use of D(2)O as a solvent, which would extend the lifetime of singlet oxygen, suggested that this species is involved in the formation of 8-oxodGuo by moxifloxacin and ciprofloxacin, but not by lomefloxacin and BAY y3118. Similarly, it was found that singlet oxygen was not involved in strand break formation. Thus, the evidence suggests that fluoroquinolones can photochemically produce DNA damage by both type I and type II mechanisms.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Nonlinearity of nuclear enlargement in hepatocytes induced by the carcinogen N'-nitrosomorpholine in Ovo.

In this study we investigated the effects of different doses of the carcinogenic nitrosamine N'-nitrosomorpholine (NNM) on the occurrence of enlarged nuclei in embryonic turkey liver in order to evaluate whether this parameter might represent a quantitative indicator of chemically induced hepatocarcinogenesis. Therefore fertile embryo turkey eggs were injected with NNM over a dose range of 125 microg-8 mg/egg at the first day of incubation. After incubation for 24 days, the embryonic livers were removed and processed for histologic evaluation. The induction of hepatocytes with enlarged nuclei (nuclear profiles > 35 microm2 was quantitated morphometrically in hematoxylin and eosin (H&E)-stained sections. The NNM treatment increased both the number of enlarged hepatocyte nuclei and the areas of the individual profiles of the enlarged nuclei in a dose-dependent manner. Exposure to 500 microg-8 mg NNM/egg resulted in a statistically significant increase in the number of hepatocytes with enlarged nuclei. The lower doses of 250 microg and 125 microg NNM/egg showed a similar albeit not significant trend. Signs for cytotoxic effects on the hepatocytes, such as necrosis or enhanced cytoplasmic vacuolization, were observed in tissue samples of embryos exposed to 4 or 8 mg NNM, but not after treatment with lower doses. The dose-effect curve for the induction of the nuclear enlargement was nonlinear, with a moderate slope for lower dose levels of 125-500 microg/egg and a steep slope for higher dose levels of 1-8 mg. Findings in rodents indicate a pathogenic link between the occurrence of enlarged nuclei and hepatocarcinogenesis. Based on the results with NNM, it is suggested that the in ovo model may represent a rapid, convenient, and inexpensive experimental approach for dose effect investigations on chemically induced hepatocarcinogenesis.

Animals↗
PubMed1999

Preclinical safety evaluation of moxifloxacin, a novel fluoroquinolone.

The toxicity of moxifloxacin (BAY 12-8039), a novel fluoroquinolone with a broad spectrum of antibacterial activity, was evaluated in a comprehensive programme of toxicological studies that included single and multiple dose toxicity studies in rats, mice, dogs and monkeys, reproductive system toxicity studies in rats and monkeys and in-vitro and in-vivo mutagenicity assays. Although moxifloxacin is not intended for long-term clinical use, an accelerated bioassay in target organs to assess carcinogenesis was performed in rats. In addition to the routine toxicological programme required for the development of a drug intended for short-term administration, a major part of the preclinical programme for moxifloxacin comprised studies designed specifically to address the safety issues known to be features of fluoroquinolones, i.e. adverse effects on the central nervous and cardiovascular systems, phototoxicity, arthrotoxicity and oculotoxicity. The results of the toxicological investigations confirmed that the safety profile of moxifloxacin is comparable to those of other fluoroquinolones.

Animals↗
PubMed1998

Preclinical safety evaluation of cerivastatin, a novel HMG-CoA reductase inhibitor.

Cerivastatin is a new but structurally distinct 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor ("statin"). It effectively decreases low-density lipoprotein (LDL) cholesterol at 1% of the doses of other currently available statins. The toxicology of cerivastatin was evaluated in a comprehensive program of studies including: (1) single- and multiple-dose toxicity studies in rats, mice, minipigs, dogs, and monkeys; (2) reproductive toxicity studies in rats and rabbits; (3) in vitro and in vivo mutagenicity assays in rats and mice; and (4) carcinogenicity studies in rats and mice. In addition, studies were undertaken to investigate the effects of cerivastatin on lens opacity, testicular tissue, and hemorrhage in dogs. Oral administration of single and multiple doses of cerivastatin over periods ranging from 4 weeks to 24 months was generally well tolerated. Adverse effects were similar to those observed with other statins and primarily involved the liver and muscle tissue. At the high doses used in the toxicologic studies, cerivastatin caused elevations in serum transaminases and creatine phosphokinase levels as well as some degeneration of muscle fibers in rats, mice, dogs, and minipigs. In dogs, the species most sensitive to statins, cerivastatin caused erosions and hemorrhages in the gastrointestinal tract, bleeding in the brain stem with fibroid degeneration of vessel walls in the choroid plexus, and lens opacity. Apart from minor morphologic changes in the testicular tissue of dogs--the only organ for which a comparably low margin of safety was observed--cerivastatin had no significant effects on the male or female reproductive system. Cerivastatin also caused no primary embryotoxic or teratogenic effects. With the exception of cerivastatin-induced effects on the eyes and testicles, administration of mevalonic acid reversed the toxicologic effects of cerivastatin, indicating that the toxic effects were related to its mode of action and not to any intrinsic toxicity of the molecule itself. There was no evidence that cerivastatin had any mutagenic effects and, in contrast to other statins, high doses of cerivastatin did not induce tumors in rats. The main metabolite of cerivastatin was well tolerated systemically in all animals, including dogs. Overall, cerivastatin has a similar toxicologic profile to other statins and is a well-tolerated HMG-CoA reductase inhibitor.

Administration, Oral↗
PubMed1998

Calcium channel blockers and the risk of cancer: a preclinical assessment.

The preclinical evidence for a potential influence of calcium channel blockers (CCBs) on carcinogenesis is discussed in the light of a broad database from rodent carcinogenicity studies as well as literature data. In all bioassays performed in rats and mice on the dihydropyridine CCBs--nifedipine, nimodipine, nisoldipine, and nitrendipine--no evidence was found for a carcinogenic potential of these compounds. Calcium is an essential intracellular signal for cell proliferation and apoptosis. The crucial role of increased cell proliferation in all stages of carcinogenesis is well documented. Some indirect experimental evidence also points to a role of defective apoptosis in tumor promotion. CCBs uniformly inhibit cell proliferation, whereas the influence of CCBs on apoptosis is inconsistent, resulting in an inhibition or increase in apoptosis dependent on cell type. Accordingly, antitumorigenic effects of CCBs have been reported based on their antiproliferative action. A tumor-promoting effect of CCBs based on inhibition of apoptosis, however, remains purely speculative and, in fact, can be denied based on the results of in vivo bioassays. It is therefore concluded that there is no preclinical evidence that should give rise to concern over the carcinogenic potential of dihydropyridine-type CCBs.

Animals↗
PubMed1998

Determination of the excitatory potencies of fluoroquinolones in the central nervous system by an in vitro model.

Fluoroquinolones have been reported to induce central nervous system side effects, including seizures and psychiatric events. Although relatively rare in patients up to now, the proconvulsant activity depends on the chemical structure and might be a critical endpoint of some new representatives of this valuable class of antimicrobials. The electrophysiological determination of field potentials in the CA1 region of the rat hippocampus slice allowed an assessment of the excitatory potential of fluoroquinolones and might be predictive for their neurotoxic potency in vivo. An optimization of this method and its extension to other fluoroquinolones resulted in a defined rank order. Well-known already-marketed quinolones as well as some fluoroquinolones under evaluation and development were used. The dose range tested was between 0.5 and 4 mumol/liter, which was comparable to the therapeutic concentration in the brain. All tested compounds increased the population spike amplitude in a concentration-dependent manner, and the resulting excitatory potency was highly dependent on the chemical structure, with compounds ranging from least to most excitatory as follows: ofloxacin, ciprofloxacin, nalidixic acid, moxifloxacin, BAY x 8843, [corrected] fleroxacin, lomefloxacin, enoxacin, clinafloxacin (much more excitatory than enoxacin), tosufloxacin, trovafloxacin, BAY 15-7828, and BAY x 9181 (much more excitatory than BAY 15-7828). The proposed hippocampus slice model not only is suitable for giving valuable alerts as to convulsive potential during candidate selection but also enables mechanistic investigations. These investigations pointed to the N-methyl-D-aspartate receptor as the probable target of the fluoroquinolone effects.

Analysis of Variance↗
PubMed1997

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Ammonium Sulfate↗
PubMed1997

Quinolone antibiotic photodynamic production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured liver epithelial cells.

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lomefloxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50-400 microM) or up to 3.6-fold in the presence of CFX (50-400 microM) and UVA (20 J/cm2) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm2. Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.

8-Hydroxy-2'-Deoxyguanosine↗