Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum

Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.     

Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum

We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.     

Recovery of Colony-Forming Ability and Genetic Marker Activity by UV-Damaged Hemophilus influenzae

The rate of recovery of UV-irradiated Hemophilus influenzae from acriflavine-sensitized loss of colony-forming ability was studied at various acriflavine concentrations, UV doses, and temperatures. This rate (as calculated from an equation based upon certain assumptions) was on the order of 0.07 per minute per cell at 37°C. This did not vary greatly with UV dose or acriflavine concentration, but did with temperature, giving a ΔH‡ of about 16 kcal/mole. In another set of experiments, cells bearing two genetic markers (resistance to 2000 μg/ml streptomycin and to 2.5 μg/ml novobiocin) were irradiated and then incubated without acriflavine. DNA extracts made from samples taken after various periods of incubation time were assayed on antibiotic-sensitive cells using acriflavine to inhibit repair during and following transformation. It was found that both in vivo irradiated markers were reactivated in the donor to approximately the same extent (with a rate constant of 0.04 per minute). This result was in contrast to the results obtained when extracted DNA bearing the same markers was irradiated in vitro and used to transform cells. In this latter case the streptomycin marker was much more sensitive than the novobiocin marker. This difference is interpreted as being due to the mechanics of the transformation system.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

In vivo nicking and rejoining of nuclear DNA in ultraviolet-irradiated radiation-resistant and sensitive strains of Dictyostelium discoideum

Summary Some aspects of DNA repair in several radiation-resistant and radiation-sensitive strains of Dictyostelium discoideum were investigated by using alkaline sucrose gradients to analyze for the production and resealing of single-strand breaks following irradiation with 254 nm UV. All radiation-resistant strains and all mutants assayed that are sensitive to both UV and ⁶⁰Co gamma rays produced singlestrand breaks in their nuclear DNA after a UV fluence of 15 J/m². Mutants at the radC locus which are sensitive to UV but as resistant as their parental strains to ⁶⁰Co gamma rays produced many fewer single-strand breaks in their DNA after irradiation with UV. Thus, the radC mutations alter a repair pathway specific for UV-induced DNA damage and presumably affect the activity of a UV-damage-specific endonuclease involved in excision repair. All radiation-resistant strains and all of our mutants sensitive to gamma rays rejoined much of their DNA during a three-hour post-UV-irradiation incubation, suggesting that these strains have at least a partially intact excision repair system.Abbreviations used UV ultraviolet light - PBS phosphate buffered saline - cpm counts per minute     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.