SARS-CoV-2 seroprevalence and determinants of infection in young adults: a population-based seroepidemiological study.

OBJECTIVES: Most SARS-CoV-2 seroprevalence studies have focussed on adults and high-risk populations, and little is known about young adults. The objective of the present study was to provide evidence on the SARS-CoV-2 seroprevalence among young adults in Germany and to explore determinants associated with seropositivity in general and, specifically, with previously undetected infections. STUDY DESIGN: This was a population-based SARS-CoV-2 seroprevalence study. METHODS: In November 2020, a population-based study on SARS-CoV-2 seroprevalence in young adults (aged 18-30 years) was conducted in a large German city. Serum samples were obtained to analyse the SARS-CoV-2 antibody status using the Elecsys Anti-SARS-CoV-2 immunoassay. Descriptive statistics and odds ratios (ORs) of seropositivity and of previously undetected infections in relation to different determinants were calculated. RESULTS: Among 2186 participants, SARS-CoV-2 antibodies were detected in 72 individuals, equalling a test performance-adjusted seroprevalence of 3.1% (95% confidence interval [CI]: 2.4-4.0). Based on reported COVID-19 cases to the public health authority, a moderate underascertainment rate of 1.7 was calculated. Seropositivity was higher among individuals who sought COVID-19-related information from social media (OR: 1.83, 95% CI: 1.2-3.1), and undetected COVID-19 infections were more prevalent among men and those not adhering to social distancing. CONCLUSIONS: The results show a substantial underascertainment of SARS-CoV-2 infections among young adults and indicate that seroprevalence is likely to be much higher than the reported COVID-19 prevalence based on confirmed COVID-19 cases in Germany. Preventive efforts should consider the heterogeneity of risk profiles among the young adult population.

Underascertainment of COVID-19 cases among first responders: a seroepidemiological study.

BACKGROUND: Providing frontline support places first responders at a high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. AIMS: This study was aimed to determine the anti-SARS-CoV-2 seroprevalence in a cohort of first responders (i.e. firefighters/paramedics), to detect the underascertainment rate and to assess risk factors associated with seropositivity. METHODS: We conducted a serological survey among 745 first responders in Germany during 27 November and 4 December 2020 to determine the anti-SARS-CoV-2 seroprevalence using Elecsys® Anti-SARS-CoV-2 immunoassay (Roche Diagnostics, Mannheim, Germany). As part of the examination, participants were asked to provide information on coronavirus disease 2019 (COVID-19)-like-symptoms, information on sociodemographic characteristics and workplace risk factors for a SARS-CoV-2 infection and any prior COVID-19 infection. Descriptive statistics and logistic regression analysis were performed and seroprevalence estimates were adjusted for test sensitivity and specificity. RESULTS: The test-adjusted seroprevalence was 4% (95% CI 3.1-6.2) and the underascertainment rate was 2.3. Of those tested SARS-CoV-2 antibody positive, 41% were aware that they had been infected in the past. Seropositivity was elevated among paramedics who worked in the emergency rescue team providing first level of pre-hospital emergency care (6% [95% CI 3.4-8.6]) and those directly exposed to a COVID-19 case (5% [95% CI 3.5-8.1]). Overall, the seroprevalence and the underascertainment rate were higher among first responders than among the general population. CONCLUSIONS: The high seroprevalence and underascertainment rate highlight the need to mitigate potential transmission within and between first responders and patients. Workplace control measures such as increased and regular COVID-19-testing and the prompt vaccination of all personnel are necessary.

Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities.

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10-15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (alpha) or reticular (beta) nuclear patterns throughout interphase. In contrast to topoisomerase IIalpha, topoisomerase IIbeta was completely excluded from nucleoli. In mitosis, topoisomerase IIbeta diffused completely into the cytosol, whereas topoisomerases I and IIalpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIalpha accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2-3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIalpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIalpha, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIbeta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIbeta is released from the heterochromatin, whereas topoisomerase I and IIalpha remain chromosome bound. Scaffold-associated topoisomerase IIalpha appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

Characterization of novel human leukemic cell lines selected for resistance to merbarone, a catalytic inhibitor of DNA topoisomerase II.

Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo IIalpha, increased topo IIbeta, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo IIalpha may play a role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo IIalpha. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistances to topo II and topo I inhibitors.

Ultra-short laser-accelerated proton pulses have similar DNA-damaging effectiveness but produce less immediate nitroxidative stress than conventional proton beams.

Ultra-short proton pulses originating from laser-plasma accelerators can provide instantaneous dose rates at least 10(7)-fold in excess of conventional, continuous proton beams. The impact of such extremely high proton dose rates on A549 human lung cancer cells was compared with conventionally accelerated protons and 90 keV X-rays. Between 0.2 and 2 Gy, the yield of DNA double strand breaks (foci of phosphorylated histone H2AX) was not significantly different between the two proton sources or proton irradiation and X-rays. Protein nitroxidation after 1 h judged by 3-nitrotyrosine generation was 2.5 and 5-fold higher in response to conventionally accelerated protons compared to laser-driven protons and X-rays, respectively. This difference was significant (p < 0.01) between 0.25 and 1 Gy. In conclusion, ultra-short proton pulses originating from laser-plasma accelerators have a similar DNA damaging potential as conventional proton beams, while inducing less immediate nitroxidative stress, which probably entails a distinct therapeutic potential.

Modulation of beta1-adrenoceptor activity by domain-specific antibodies and heart failure-associated autoantibodies.

OBJECTIVES: Our study attempted to gain further understanding of the allosteric effects of human autoantibodies on beta1-adrenergic receptor (beta1-AR) function. BACKGROUND: Recently, we reported on the existence of activating anti-beta1-AR antibodies in patients with dilated cardiomyopathy (DCM 26% prevalence) or ischemic cardiomyopathy (ICM, 10% prevalence); however, their functional effects have not yet been thoroughly characterized. METHODS: In this study we detected functionally active receptor-antibodies in 8 out of 30 DCM patients. Their immunological and functional properties were analyzed using both synthetic receptor-peptides and intact recombinant human beta1-AR, and were compared with those of heterologous antibodies to selected beta1-AR domains generated in rabbits and mice. RESULTS: Rabbit, mouse, and human anti-beta1-AR against the second extracellular domain preferentially bound to a native receptor conformation and impaired radioligand binding to the receptor. However, their functional effects differed considerably: Rabbit and mouse antibodies decreased both basal and agonist-stimulated cAMP production, whereas the patient antibodies (n = 8) increased basal, and six of them also increased agonist-stimulated receptor activity (i.e., acted as receptor-sensitizing agents). Two out of eight human anti-beta1-AR increased basal but decreased agonist-stimulated receptor activity (i.e., acted as partial agonists). CONCLUSIONS: Antibodies against the same small beta1-AR domain can have very divergent allosteric effects, ranging from inhibitory to agonist-promoting activities. Activating autoantibodies were associated with severe cardiac dysfunction and thus might be involved in the development and/or course of human cardiomyopathy.

Activating beta-1-adrenoceptor antibodies are not associated with cardiomyopathies secondary to valvular or hypertensive heart disease.

OBJECTIVES: We investigated whether autoantibodies against the human beta-adrenergic receptor (beta-AR) might be involved in cardiomyopathies secondary to valvular heart disease (VHD) or hypertensive heart disease (HHD). BACKGROUND: Autoimmunity to beta-AR has been proposed as a pathogenic principle in human cardiomyopathy. Recently, by the use of intact recombinant human beta-AR, we were able to confirm the existence of functionally active anti-beta-1-AR autoantibodies in patients with dilated cardiomyopathy (26% prevalence) or ischemic cardiomyopathy (10% prevalence); however, their prevalence in other (secondary) cardiomyopathies remained to be determined. METHODS: Immunoglobulin G (IgG) was prepared from the sera of 28 VHD and 19 HHD patients and first screened by a peptide-based enzyme-linked immunosorbent assay (antigens: aminoterminus, second extracellular loop [ECII] and carboxyterminus of human beta-1- and beta-2-AR). IgG from 108 gender- and age-matched healthy subjects served to define the threshold for positive immunoreactions. Positive sera were further screened for their ability to recognize and activate native human beta-AR situated in a cell membrane. RESULTS: Twenty-five percent (VHD) or 11% (HHD) of the patients and 4% of the healthy controls had IgG antibodies randomly directed against all the three domains tested and both beta-AR subtypes. Only one patient with aortic valve and concomitant coronary heart disease and one healthy subject had functionally active anti-b1-AR (targeting beta-1-ECII). Moreover, one HHD patient with concomitant collagenosis had IgG that was cross-reacting with recombinant beta-AR in immunological assays but was unable to affect receptor function. CONCLUSIONS: Autoimmune reactions against the human beta-AR do not appear to be associated with cardiomyopathies secondary to VHD or HHD.

Purification and functional characterization of the human beta 2-adrenergic receptor produced in baculovirus-infected insect cells.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

Alteration of topoisomerase II action is a possible molecular mechanism of HL-60 cell differentiation.

The inhibition of differentiation and persistence of proliferation in cell transformation is probably not only caused by the mutation of single genes. An additional mechanism of transcriptional control, not only of single genes but of gene programs, is possibly the alteration of the topoisomerases. These enzymes regulate the conformation of DNA by twisting and unwinding the double strands. As has been shown previously, only the genes located in relaxed DNA areas are transcribed and, therefore, the topoisomerases can be described as a gene regulation device. We present the hypothesis that topoisomerase II action is not only altered in, but also necessary for, HL-60 granulocytic cell differentiation. Thus, alteration of topoisomerases may well be a molecular mechanism of cellular differentiation.

Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies.

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [3H]CGP 12 177. Affinity purified antibodies were used for detecting the beta 1- or the beta 2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Probing human beta1- and beta2 -adrenoceptors with domain-specific fusion protein antibodies.

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta1- or beta2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta1- or beta2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [3H]CGP 12 177. Affinity purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying beta-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.

Different functional states of topoisomerase II can be discriminated by orthovanadate sensitivity in multi-drug resistant human leukemic cells.

We have studied the functional properties of topoisomerase II (Topo II) in a subclone of the HL-60 cell line, which is highly resistant to cytotoxic Topo II inhibitors, but does not express p-glycoprotein. The cells contain the two forms of human topo II with Mr 170 and 180 kDa in equal proportions. Two different states of both forms of the enzymes can be separated by anion-exchange chromatography and functionally discriminated on the basis of orthovanadate sensitivity. The EC50 of orthovanadate was 0.2 microM for the early eluting and 30 microM for the late eluting Topo II.

Nuclear Topoisomerase II Activity Changes During HL-60 Leukemic Cell Differentiation: Alterations in Drug Sensitivity and pH Dependency.

We have studied the effect of dimethyl-sulfoxide(DMSO)-induced granulocytic differentiation on the sensitivity of HL-60 cells to various cytotoxic topoisomerase II inhibitors: (i) undifferentiated HL-60 cells are highly sensitive to etoposide, while differentiated HL-60 cells are 700-1000 fold resistant to etoposide, (ii) undifferentiated HL-60 are 50-100 fold resistant against 4-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) when compared to the differentiated HL-60 cells, (iii) the addition of mAMSA to the medium inhibits granulocytic differentiation of HL-60 cells. This change in resistance pattern is probably due to an alteration of topoisomerases since distinctive iso-activites of topoisomerase can be discriminated on the basis of the pH profile, which alters markedly during differentiation. In an etoposide-resistant HL-60 cell line we found a reduced topoisomerase activity at pH 7.8/7.9. This topoisomerase iso-activity is obviously involved in etoposide cytotoxicity.

Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells.

The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

Topoisomerase II function detected as protein-DNA complexes in two sublines of HL-60 cells.

Topoisomerase II forms a cleavable complex with DNA as an intermediate in the topoisomerization reaction. Denaturation produces covalent linkage between the enzyme and DNA, and produces double-stranded DNA breaks at the site of attachment. Thereby topoisomerase function can be detected by measuring the protein-DNA complexes. A whole cell filter binding assay exploiting this mechanism is described. m-AMSA-resistant HL-60 cells have higher protein-DNA complex formation than m-AMSA-sensitive HL-60 cells, suggesting different activities of topoisomerase II in drug-resistant vs. drug-sensitive cells.

Correlation between the DNA-binding affinity of topoisomerase inhibiting drugs and their capacity to induce hematopoetic cell differentiation.

There is accumulating evidence that both type I and type II DNA-topoisomerases play a key role in cellular differentiation. Human HL-60 leukemia cells can be induced to monocytic or granulocytic differentiation with various compounds; amongst them camptothecin, a topoisomerase I inhibitor and VP-16, VM-26 and mitoxantrone, all potent topoisomerase II inhibitors. During HL-60 cell differentiation topoisomerase I activity increases and topoisomerase II activity decreases. The two isoenzymes topoisomerase II alpha and topoisomerase II beta seem to have different physiological functions in highly proliferating cells, G0 cells and differentiated cells as their expression is regulated differently. In concentrations sublethal to undifferentiated cells, m-AMSA, also a potent topoisomerase II inhibitor, is able to prevent DMSO-induced granulocytic HL-60 cell differentiation. In drug-sensitive cells derived from several sources (mouse erythroleukemia, human gastric carcinoma, human leukemia), we found a functional heterogeneity of topoisomerase activity which is altered specifically during cellular differentiation. The isoactivities can be separated by their different pH and salt requirements and they exhibit different sensitivity to topoisomerase II inhibiting drugs. Functional heterogeneity of topoisomerases seems to be a prerequisite to high drug sensitivity of the cells, since drug-resistant sublines in our experiments do not exhibit this heterogeneity. We propose that the topoisomerase II inhibiting drugs which are able to induce differentiation, namely the epipodophyllotoxines VP-16 and VM-26, inhibit subfractions of the topoisomerase II pool which are necessary to maintain the undifferentiated status of the cells. These drugs induce differentiation in concentrations 10-100-fold below the lethal dose, the concentration must be sufficient to inhibit topoisomerase II but well below the concentration to induce the cleavable complex. This might be the reason that anthracyclines with a high DNA binding affinity have low differentiation-inducing capacity.

Simultaneous presence of terminal adenylyl, cytidylyl, guanylyl, and uridylyl transferase in healthy tomato leaf tissue: separation from RNA-dependent RNA polymerase and characterization of the terminal transferases.

The presence of terminal nucleotidyl transferase activities catalyzing the addition of AMP, CMP, GMP, and UMP residues to the 3' ends of oligonucleotide primers was detected in healthy tomato plants. These enzyme activities copurify with RNA-dependent RNA polymerase during the initial stages of purification. Their separation from RNA-dependent RNA polymerase is finally achieved by DEAE chromatography: terminal transferase activities are retained on DEAE while RNA-dependent RNA polymerase does not bind in the presence of 20 mM MgCl2. Elution by a linear gradient of 0 to 400 mM NH4Cl releases all four terminal transferase activities from the DEAE column at a concentration of 270 mM NH4Cl, thus suggesting that they may belong to one enzyme molecule; this question, however, needs further clarification. The enzyme activities are completely dependent on the presence of an RNA primer and are strongly influenced by its base composition as well as its chain length. Characterization of the respective reaction products by electrophoresis on 15% polyacrylamide sequencing gels reveals striking differences as to the number of nucleotides added to a given primer. In the case of UMP transfer to U8 or A8 and in the case of GMP transfer to A8 only 1 to 6 nucleoside monophosphates are added to the 3' terminus of the oligonucleotide primer, whereas in the case of AMP transfer to A8 or U8, the CMP transfer to A8, and the GMP transfer to U8, longer chains of minimally 30 nucleotides are added to the respective primer. After gradient elution from DEAE the transferase preparation displays no nucleolytic activity when incubated in the presence of 3H-labelled ribosomal RNA or [3H]poly(A) X poly(U). Only in the case of [3H]poly(A) and [3H]poly(U) or [3H]poly(C) 10 to 15% of the radioactivity is transferred to acid-soluble counts.

In vitro transcription of viroid RNA into full-length copies by RNA-dependent RNA polymerase from healthy tomato leaf tissue.

RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of full-length RNA copies of the PSTV genome. Viroid transcription requires the presence of Mn2+ and /or Mg2+ ions and is not inhibited by concentrations of 10(-5) M alpha-amanitin. This is the first report of a well-defined product synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants.

Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase from Escherichia coli.

The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, from Escherichia coli. In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication. The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

DNA topoisomerases in therapy--tenth conference. 6-8 October 1999, Amsterdam, The Netherlands.

The meeting covered basic research on DNA topoisomerases and aspects of DNA topoisomerase-directed therapy, which will be the main topic of this report. In terms of cancer therapy, the focus of the meeting was clearly on camptothecins (CPTs) and related compounds, that stabilize covalent DNA intermediates of topoisomerase I. Results were presented showing that these drugs might act in a tumor-specific manner because tumor cells have defects in degradation pathways of DNA-linked topoisomerase I. On the other hand, a DNA-tyrosine phosphodiesterase has been discovered, which removes topoisomerase I from its covalent DNA-linkage and thus might be a new mechanism of drug resistance. Reports on recent clinical trials of first-generation water soluble CPT analogs (topotecan; SmithKline Beecham, and irinotecan; Yakult Honsha KK), confirmed earlier findings that these drugs have major limitations due to the half-life of the active lactone form and other pharmacokinetic factors, resulting in a major schedule dependency of the toxicity. Solutions to that problem will possibly come from an oral application regimen or liposomal packaging of the drugs. Several new CPT analogs at preclinical stages of development might also improve on these problems by providing a greater stability of the lactone ring, higher DNA-binding affinity, and reduced water solubility. New drugs might be developed from a number of new non-CPT compounds, which inhibit the activity of DNA topoisomerases, but do not stabilize the DNA-linked form of the enzymes. Some of these compounds display reasonable preclinical anticancer activity. A second focus of the meeting was on therapeutic targeting of microbial DNA topoisomerases. On the one hand, the antibiotic potential of the quinolones has been extended to Gram-positive pathogens, particularly Streptococcus pneumoniae. On the other hand, cloning and biochemical characterization of the DNA topoisomerases of eukaryotic parasites, such as Plasmodium falciparum or Candida albicans, have been completed and the search for specific inhibitors targeting these enzymes are under way.