Delta-type DNA polymerase characterized from Drosophila melanogaster embryos.

Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases.     

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

Evaluation of a rule base for identifying contact allergens by using a regulatory database: Comparison of data on chemicals notified in the European Union with "structural alerts" used in the DEREK expert system

To assess the suitability of the use of structural alerts to identify the skin-sensitising properties of chemicals, the 40 originally published structural alerts for the prediction of skin-sensitisation properties used by the DEREK system (which now contains 59 structural alerts), have been evaluated against a database developed in the German Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), which contains data submitted under the procedure for notifying new chemicals within the European Union. The evaluation of the 40 structural alerts used in DEREK revealed that eight of the 40 structural alerts for the prediction of skin-sensitising potential could be used without any further refinement. Ten structural alerts may need further specifications or refinements in order avoid false-positive predictions - proposals for refinement are discussed. Not enough substances were found within the BgVV database (containing data for more than 1000 substances) to evaluate ten of the DEREK substructures; hence, for these structural alerts, a judgement on their suitability for prediction of skin-sensitisation properties in expert systems is not possible. For 12 structural alerts, no comparative result could be obtained, because these rules did not "fire" for any of the examined chemicals. As a general result of the evaluation process, the approach of using structural alerts for the prediction of skin-sensitising properties of chemicals proved to be reliable. Proposals are given for a refinement of the structural alerts for prediction of contact allergy used in the DEREK system. In addition, advice and several preconditions are given, that apply when trying to teach a computer system to use structural alerts to predict toxicological properties.     

Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Creation of fundamental rules for a decision support system

The notification procedure of the European Union (EU) for new chemicals requires the application of protocols on physicochemical and toxicological tests for the evaluation of physicochemical properties and probable toxic effects of each notified substance. A computerised database was developed from data sets and toxicological test protocols relating to substance properties responsible for skin and eye irritation/corrosion. To develop specific structure-activity relationship (SAR) models and to find rules for a decision support system (DSS) to predict local irritation/corrosion, physical property data, chemical structure data and toxicological data for approximately 1300 chemicals, each having a purity of 95% or more, were evaluated. The evaluation demonstrated that the lipid solubility and aqueous solubility of a chemical are relevant to, or - in some cases - responsible for, the observed local effects of a substance on the skins and eyes of rabbits. The octanol/water partition coefficient and the measured value of the surface tension of a saturated aqueous solution of the substance give additional information that permits the definition of detailed SAR algorithms that use measured solubility values. Data on melting points and vapour pressure can be used to assess the intensity and duration of local contact with a chemical. Considerations relating to the reactivity of a pure chemical can be based on molecular weight and the nature of the heteroatoms present. With respect to local lesions produced following contact with the skin and eyes of rabbits, the data evaluation revealed that no general "local irritation/corrosion potential" of a chemical can be defined. A variety of mechanisms are responsible for the formation of local lesions on the skin or in the eyes: serious lesions are produced by mechanisms different from those that cause moderate irritation in these organs. In order to develop a DSS that uses the information extracted from the database, chemical main groups were categorised on the basis of their empirical formulae, and rules were defined of the type IF (physicochemical property) A, THEN not (toxic) effect B, based on correlations between specific local effects and measured physicochemical values. Other rules of the type IF substructure A, THEN effect B were developed based on correlations between specific local effects and the submitted structural formulae. Reactive chemical substructures relevant to the formation of local lesions and rules for the prediction of the absence of any skin irritation potential were identified. Proposals are made relating to the development of alternatives to eye irritation testing with rabbits.