Seroprevalences of antibodies against ToRCH infectious pathogens in women of childbearing age residing in Brazil, Mexico, Germany, Poland, Turkey and China.

Determination of antibodies against ToRCH antigens at the beginning of pregnancy allows assessment of both the maternal immune status and the risks to an adverse pregnancy outcome. Age-standardised seroprevalences were determined in sera from 1009 women of childbearing age residing in Mexico, Brazil, Germany, Poland, Turkey or China using a multiparametric immunoblot containing antigen substrates for antibodies against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), herpes simplex viruses (HSV-1, HSV-2), Bordetella pertussis, Chlamydia trachomatis, parvovirus B19, Treponema pallidum and varicella zoster virus (VZV). Seroprevalences for antibodies against HSV-1 were >90% in samples from Brazil and Turkey, whereas the other four countries showed lower mean age-adjusted seroprevalences (range: 62.5-87.9%). Samples from Brazilian women showed elevated seroprevalences of antibodies against HSV-2 (40.1%), C. trachomatis (46.8%) and B. pertussis (56.6%) compared to the other five countries. Seroprevalences of anti-T. gondii antibodies (0.5%) and anti-parvovirus B19 antibodies (7.5%) were low in samples from Chinese women, compared to the other five countries. Samples from German women revealed a low age-standardised seroprevalence of anti-CMV antibodies (28.8%) compared to the other five countries. These global differences in immune status of women in childbearing age advocate country-specific prophylaxis strategies to avoid infection with ToRCH pathogens.

A 3-day short course of palifermin before HDT reduces toxicity and need for supportive care after autologous blood stem-cell transplantation in patients with multiple myeloma.

BACKGROUND: We retrospectively determined whether a 3-day short course of palifermin could reduce the toxicity of high-dose therapy (HDT) and autologous blood stem-cell transplantation (ASCT) in patients with multiple myeloma (MM). PATIENTS AND METHODS: Sixty-seven consecutive patients received 60 mug/kg palifermin for 3 days before HDT with melphalan 200 or 140 mg/m(2) for patients with renal failure (group A). Granulocyte colony-stimulating factor (G-CSF) was applied after ASCT. Data on haematopoietic reconstitution and toxicity were compared with two previously published patient groups from our institution who had received pegfilgrastim but not palifermin (group B, n = 21) and patients who had received neither palifermin nor G-CSF (group C, n = 21). RESULTS: In group A, patients with renal failure had a significantly higher risk for severe mucositis (64% versus 16%, P < 0.002). Patients with normal renal function who received palifermin experienced significantly less days of hospitalisation (P < 0.05) and less need for narcotic analgesia (P < 0.05), parenteral nutrition (P < 0.05) and erythrocyte transfusions (P < 0.05) in comparison with groups B and C. Time to haematopoietic reconstitution was not compromised by the use of palifermin. CONCLUSIONS: In conclusion, a short 3-day course of palifermin may be able to reduce the toxicity of HDT and ASCT in patients with MM. Patients with impaired renal function at the time of HDT need additional strategies to further reduce the incidence of severe mucositis.

[Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood].

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of patients with breast tumors and healthy controls. Frequency of RASSF1A, Cyclin D2 and RARbeta2 methylation was detected using methylation-specific PCR in the extracellular DNA, extracted from plasma and cell-surface bound fractions of patient blood. Methylation of at least one of these genes was found in plasma of 13% patients with benign breast fibroadenoma and in 60% of breast cancer patients. Using cell-surface bound DNA as a substrate for PCR have lead to increase of gene methylation detection frequency up to 87% in fibroadenoma and 95% in breast cancer patients without false positive controls. GAPDH, RASSF8, Ki-67 RNA and 18S RNA were quantified using RT-qPCR of the extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The main part of the extracellular RNA was shown to be cell-surface bound. Results show a higher amount of RASSF8, Ki-67 RNA and 18S RNA in plasma and cell-bound fraction of patients with breast cancer compared with patients with benign tumors and healthy controls. The data indicate that the specific RNA quantification in blood plasma is valuable for discrimination between cancer and benign tumors, which can be detected with high sensitivity using analysis of methylated RASSF1A, Cyclin D2 and RARbeta2 genes in extracellular circulating DNA.

The large form of ADAR 1 is responsible for enhanced hepatitis delta virus RNA editing in interferon-alpha-stimulated host cells.

Hepatitis delta virus (HDV) RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore indirectly regulates HDV replication. Editing is thought to be catalysed by the adenosine deaminase acting on RNA1 (ADAR1) of which two different forms exist, interferon (IFN)-alpha-inducible ADAR1-L and constitutively expressed ADAR1-S. ADAR1-L is hypothesized to be a part of the innate cellular immune system, responsible for deaminating adenosines in viral dsRNAs. We examined the influence of both forms on HDV RNA editing in IFN-alpha-stimulated and unstimulated hepatoma cells. For gene silencing, an antisense oligodeoxyribonucleotide against a common sequence of both forms of ADAR1 and another one specific for ADAR1-L alone were used. IFN-alpha treatment of host cells led to approximately twofold increase of RNA editing compared with unstimulated controls. If ADAR1-L expression was inhibited, this substantial increase in editing could no longer be observed. In unstimulated cells, ADAR1-L suppression had only minor effects on editing. Inhibition of both forms of ADAR1 simultaneously led to a substantial decrease of edited RNA independently of IFN-alpha-stimulation. In conclusion, the two forms of ADAR1 are responsible almost alone for HDV editing. In unstimulated cells, ADAR1-S is the main editing activity. The increase of edited RNA under IFN-alpha-stimulation is because of induction of ADAR1-L, showing for the first time that this IFN-inducible protein is involved in the base modification of replicating HDV RNA. Thus, induction of ADAR1-L may at least partially cause the antiviral effect of IFN-alpha in natural immune response to HDV as well as in case of therapeutic administration of IFN.

Measurement of chemopreventive efficacy in skin biopsies.

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY.

Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.

The 5'-proximal hairpin loop of lbi RNA is a key structural element in repression of D-galactan II biosynthesis in Klebsiella pneumoniae serotype O1.

The lbi (lipopolysaccharide biosynthesis interfering) RNA of phage Acm1, an untranslated RNA transcript of 97 nucleotides, previously shown to affect O-polysaccharide biosynthesis in various Escherichia coli strains, was found to downregulate the synthesis of the D-galactan II component of the O-specific polysaccharide in Klebsiella pneumoniae serotype O1. Enzymatic and Pb2+ probing experiments revealed that lbi RNA consists of two consecutive stem-loop structures, the 5'-proximal hairpin loop of 15 nucleotides being particularly accessible to single strand-specific probes. Based on the assumption that the 5'-proximal hairpin loop may be involved in an antisense interaction with cellular target RNAs, we randomly mutagenized one or two of its central nucleotides. Expression of mutated lbi RNA variants in K. pneumoniae serotype O1 relieved at least partly the repression of D-galactan II formation. In addition, a truncated version of lbi RNA lacking the 3'-proximal hairpin loop was almost as efficient as the wild-type RNA in downregulating D-galactan II synthesis. The results obtained indicate that the 5'-proximal hairpin loop of lbi RNA functions as a key structural element in the mechanism leading to the inhibition of D-galactan II biosynthesis in K. pneumoniae serotype O1.

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage.

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3'-OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'- S -phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3'- S -phosphorothiolate-modified ptRNA carrying a 7 nt 5'-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5'-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn(2+)or Cd(2+). To suppress aberrant cleavage, we also constructed a 3'- S -phosphorothiolate-modified ptRNA with a 1 nt 5'-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3'- S -phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.

Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis.

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a >/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.

Antibacterial and sebosuppressive efficacy of a combination of chloramphenicol and pale sulfonated shale oil. Multicentre, randomized, vehicle-controlled, double-blind study on 91 acne patients with acne papulopustulosa (Plewig and Kligman's grade II-III).

In a 3-armed, multicentre, randomized, double-blind, vehicle-controlled study involving 91 patients with acne papulopustulosa, Plewig's grade II-III, evidence could be provided of a significant reduction of the propionibacteria as well as a subosuppressive effect (squalene reduction) under a combination of 1% chloramphenicol (CAS 56-75-7) and 0.5% pale sulfonated shale oil versus the alcoholic vehicle (1-2 ml twice daily). Likewise, monotherapy with chloramphenicol resulted in a significant reduction in bacteria compared to the vehicle. The combination therapy was superior to the monotherapy with regard to the sebosuppressive effects. Based on a kinetics test carried out for a total of 2 h, a clinically relevant percutaneous absorption of chloramphenicol was ruled out. The chloramphenicol serum level was between < 5.0 microgram/l to 180 microgram/l (average 25 micogram/l). This is important because with systemic application (peroral, i.v.), the therapeutic chloramphenicol level is > 25 mg/l (25,000 microgram 1). None of the blood count and serum parameters were pathologically changed in a clinically relevant way before and after the therapy. An induction of resistance against chloramphenicol in the propionibacteria could be excluded. No adverse events and side effects occurred. The topical therapy of acne papulopustulosa with chloramphenicol as a monosubstance or in combination with pale sulfonated shale oil represents an effective and safe local antibiotic treatment possibility.

Anti-inflammatory action of pale sulfonated shale oil (ICHTHYOL pale) in UVB erythema test.

OBJECTIVE AND DESIGN: In a clinical research institute the anti-inflammatory effect of pale sulfonated shale oil versus hydrocortisone was investigated in a placebo-controlled clinical trial using a double-blind design and random assignment of the treatments to the test fields. SUBJECTS: 20 male and female volunteers with healthy skin in the test areas participated in this trial. TREATMENT: All subjects received different concentrations of pale sulfonated shale oil (2% and 4%), the active ingredient-free vehicle and a reference product containing 0.5% hydrocortisone. METHODS: Approximately 300 microl of test preparation were applied to the treatment fields. The medication was administered for 23 h. An untreated, irradiated and an untreated, non-irradiated control field were included as well. The test fields were compared intraindividually. Four different UV-doses (1; 1.25; 1.6 and 2 MED) were tested in each volunteer. The test fields were occluded for 6 h. After 7 h, measurement with a colorimeter was carried out. After measurement, the treatment was repeated. The test fields were occluded for a further 16 h and test preparations carefully wiped off. One hour later, post-irradiation color measurements were made by colorimetric detection. Variance analysis was used for statistical evaluation. RESULTS: 4% pale sulfonated shale oil and 0.5% hydrocortisone had a significantly greater efficacy than the vehicle (p = 0.0001). There were no differences between the efficacy of 4% pale sulfonated shale oil and 0.5% hydrocortisone (p = 0.5169). CONCLUSIONS: These results demonstrate the anti-inflammatory efficacy of 4% pale sulfonated shale oil and help explain the clinical effects of the drug in the therapy of atopic eczema.

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference.

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of (pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for the pro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA to E. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.

Impaired coronary dilator responses to substance P and impaired flow-dependent dilator responses in heart transplant patients with graft vasculopathy.

OBJECTIVES: Because pathologic mechanisms for transplant vasculopathy are still uncertain, we tested the hypothesis that endothelial function, in terms of the release of endothelium-derived relaxing factor (EDRF), is impaired in patients with evidence of angiographic transplant vasculopathy. BACKGROUND: The long-term prognosis after heart transplantation is mainly determined by the development of transplant vasculopathy. METHODS: The study included 23 patients undergoing diagnostic cardiac catheterization approximately 40 months after heart transplantation. Patients were classified into those with (n = 8) and those without (n = 15) angiographic evidence of transplant vasculopathy. Coronary flow velocity (by intravascular Doppler echocardiography) and epicardial coronary diameter (by quantitative angiography) were determined after intracoronary bolus injections (1 ml) of the endothelium-dependent dilator substance P (20 pmol) and the endothelium-independent dilators nitroglycerin (0.1 mg) and papaverine (8 mg). Substances were injected through the lumen of the Doppler catheter, which was placed into the midportion of the left anterior descending artery. RESULTS: Increases in blood flow velocity in response to substance P were significantly less in patients with than in patients without evidence of transplant vasculopathy. In addition, flow-mediated dilation of epicardial coronary arteries in response to papaverine was abolished in patients with such evidence. Vasodilation of epicardial coronary arteries in response to nitroglycerin and increases in flow velocity in response to papaverine were similar in both groups. CONCLUSIONS: These results suggest that transplant vasculopathy in heart transplant patients is associated with endothelial dysfunction (that is, impaired EDRF-mediated vasodilation). Furthermore, responsiveness of epicardial arteries to increased flow appears to be abolished in patients with evidence of transplant vasculopathy. These abnormal vascular functions may contribute to the pathogenesis of transplant vasculopathy and its vascular complications.