High Molecular Weight Fragments Research Articles

Gauging DNA degradation among common insect trap preservatives

AbstractGenetic methods for species identification are becoming increasingly popular and can accelerate insect monitoring. However, obtaining good DNA quality and quantity from insect traps remains a challenge for field studies. Ethylene glycol, propylene glycol, and Renner solution have been previously suggested as suitable preservatives for the collection of genetic material, but a systematic overview of their performance under compromising field conditions is lacking. Here we experimentally test whether and how different preservatives affect DNA quality under different conditions and evaluate how choice of preservative may affect metabarcoding and more demanding downstream applications (e.g., RADseq). For this, we used the house cricket, Acheta domesticus (L.) (Orthoptera: Gryllidae), and tested propylene glycol, ethylene glycol, and Renner solution for their ability to preserve DNA over 27 days in various dilutions and temperatures. DNA quality was measured as DNA fragmentation and success rates in PCR amplifying a COI fragment of 658, 313, or 157 bp. Undiluted propylene glycol and ethylene glycol always retained high molecular weight DNA at room temperature. No high molecular weight DNA was preserved at 37 °C or in any dilution. Nevertheless, the COI sequence could be amplified from samples at every condition. Renner solution did not preserve high molecular weight DNA and fragmentation increased over time at 37 °C until amplification was impossible. The results suggest that propylene glycol and ethylene glycol are suitable preservatives for collecting both genetic and morphological material, but dilution or high temperatures compromise their ability to preserve high molecular weight DNA. For genomic approaches requiring high DNA quality, additional preservatives may need to be tested.

Deriving high value products from depolymerized lignin oil, aided by (bio)catalytic funneling strategies.

Lignin holds tremendous and versatile possibilities to produce value-added chemicals and high performing polymeric materials. Over the years, different cutting-edge lignin depolymerization methodologies have been developed, mainly focusing on achieving excellent yields of mono-phenolic products, some even approaching the theoretical maximum. However, due to lignin's inherent heterogeneity and recalcitrance, its depolymerization leads to relatively complex product streams, also containing dimers, and higher molecular weight fragments in substantial quantities. The subsequent chemo-catalytic valorization of these higher molecular weight streams, containing difficult-to-break, mainly C-C covalent bonds, is tremendously challenging, and has consequently received much less attention. In this minireview, we present an overview of recent advances on the development of sustainable biorefinery strategies aimed at the production of well-defined chemicals and polymeric materials, the prime focus being on depolymerized lignin oils, containing high molecular weight fractions. The key central unit operation to achieve this is (bio)catalytic funneling, which holds great potential to overcome separation and purification challenges.

Quantification of immune cell-mediated destruction of tumor cells in vitro using the RNA disruption assay.

e15017 Background: Immune checkpoint inhibitors (ICIs) are increasingly being used in the treatment of human cancers, often in combination with cytotoxic chemotherapy drugs. However, current ICIs are costly and are only effective against a fraction of patient tumours. Thus, a tool that could reliably quantify tumour response to ICIs and predict outcome post-treatment would be highly valuable. Patients with non-responding tumours could be spared the costs and toxic side-effects of the ineffective drugs and moved promptly to alternate treatments. We have observed that a variety of structurally and mechanistically distinct chemotherapy agents induce the degradation of ribosomal RNA (rRNA) into a large number of high molecular weight fragments (between the 28S and 18S rRNAs)---a phenomenon we have termed “RNA disruption”. High tumour RNA disruption during treatment, as quantified using the RNA disruption assay (RDA), has been shown to predict pathologic complete response and improved disease-free survival in breast cancer patients. Methods: To assess whether RDA could be used to monitor immune cell killing of tumour cells, we collected peripheral blood mononuclear cells (PBMCs) from consenting healthy human donors and enriched the cell population for natural killer (NK) cells to 90% using a negative selection approach. The cells, with or without IL-2 pre-treatment, were incubated with K562 cells, after which K562 cell killing was quantified over time using a standard immunocytotoxicity assay and K562 cell RNA disruption was measured using RDA. Results: Freshly isolated human NK cells when incubated with K562 chronic myeloid leukemia cells induced cell destruction and RNA disruption in K562 cells in a dose-dependent manner. Pre-incubation with IL-2 augmented both NK cell-mediated RNA disruption and NK-mediated cytotoxicity in K562 cells. Interestingly, the pattern of K562 RNA disruption fragments generated by NK cells was similar to that generated by chemotherapy drugs. Conclusions: RDA was successfully used to quantify immune cell-mediated destruction of tumour cells, raising the prospect of its possible use to monitor tumour destruction by immune cells in vivo and to predict response to ICIs (alone or in concert with cytotoxic chemotherapy drugs).

Influence of poly(I:C) variability on thermoregulation, immune responses and pregnancy outcomes in mouse models of maternal immune activation

Maternal immune activation (MIA) models that are based on administration of the viral mimetic, poly(I:C), are widely used as experimental tools to study neuronal and behavioral dysfunctions in relation to immune-mediated neurodevelopmental disorders and mental illnesses. Evidence from investigations in non-pregnant rodents suggests that different poly(I:C) products can vary in terms of their immunogenicity, even if they are obtained from the same vendor. The present study aimed at extending these findings to pregnant mice, while also controlling various poly(I:C) products for potential contamination with lipopolysaccharide (LPS). We found significant variability between different batches of poly(I:C) potassium salt obtained from the same vendor (Sigma-Aldrich) in terms of the relative amount of dsRNA fragments in the high molecular weight range (1000–6000 nucleotides long) and with regards to their effects on maternal thermoregulation and immune responses in maternal plasma, placenta and fetal brain. Batches of poly(I:C) potassium salt containing larger amounts of high molecular weight fragments induced more extensive effects on thermoregulation and immune responses compared to batches with minimal amounts of high molecular weight fragments. Consistent with these findings, poly(I:C) enriched for high molecular weight dsRNA (HMW) caused larger maternal and placental immune responses compared to low molecular weight (LMW) poly(I:C). These variable effects were unrelated to possible LPS contamination. Finally, we found marked variability between different batches of the poly(I:C) potassium salt in terms of their effects on spontaneous abortion rates. This batch-to-batch variability was confirmed by three independent research groups using distinct poly(I:C) administration protocols in mice. Taken together, the present data confirm that different poly(I:C) products can induce varying immune responses and can differentially affect maternal physiology and pregnancy outcomes. It is therefore pivotal that researchers working with poly(I:C)-based MIA models ascertain and consider the precise molecular composition and immunogenicity of the product in use. We recommend the establishment of reference databases that combine phenotype data with empirically acquired quality information, which can aid the design, implementation and interpretation of poly(I:C)-based MIA models.

Primary emissions and secondary aerosol production potential from woodstoves for residential heating: Influence of the stove technology and combustion efficiency

To reduce the influence of biomass burning on air quality, consumers are encouraged to replace their old woodstove with new and cleaner appliances. While their primary emissions have been extensively investigated, the impact of atmospheric aging on these emissions, including secondary organic aerosol (SOA) formation, remains unknown. Here, using an atmospheric smog chamber, we aim at understanding the chemical nature and quantify the emission factors of the primary organic aerosols (POA) from three types of appliances for residential heating, and to assess the influence of aging thereon. Two, old and modern, logwood stoves and one pellet burner were operated under typical conditions. Emissions from an entire burning cycle (past the start-up operation) were injected, including the smoldering and flaming phases, resulting in highly variable emission factors. The stoves emitted a significant fraction of POA (up to 80%) and black carbon. After ageing, the total mass concentration of organic aerosol (OA) increased on average by a factor of 5. For the pellet stove, flaming conditions were maintained throughout the combustion. The aerosol was dominated by black carbon (over 90% of the primary emission) and amounted to the same quantity of primary aerosol emitted by the old logwood stove. However, after ageing, the OA mass was increased by a factor of 1.7 only, thus rendering OA emissions by the pellet stove almost negligible compared to the other two stoves tested. Therefore, the pellet stove was the most reliable and least polluting appliance out of the three stoves tested. The spectral signatures of the POA and aged emissions by a High Resolution – Time of Flight – Aerosol Mass Spectrometer (Electron Ionization (EI) at 70 eV) were also investigated. The m/z 44 (CO2+) and high molecular weight fragments (m/z 115 (C9H7+), 137 (C8H9O2+), 167 (C9H11O3+) and 181 (C9H9O4+, C14H13+)) correlate with the modified combustion efficiency (MCE) allowing us to discriminate further between emissions generated from smoldering vs flaming conditions.

Formation of high-molecular-weight compounds via the heterogeneous reactions of gaseous C8–C10 n-aldehydes in the presence of atmospheric aerosol components

Abstract A laboratory study on the heterogeneous reactions of straight-chain aldehydes was performed by exposing n-octanal, nonanal, and decanal vapors to ambient aerosol particles. The aerosol and blank filters were extracted using methanol. The extracts were nebulized and the resulting compositions were examined using a high-resolution time-of-flight aerosol mass spectrometer. The mass spectral analysis showed that the exposures of the aldehydes to aerosol samples increased the peak intensities in the high mass range. The peaks in the organic mass spectra of the aerosol samples after exposure to different aldehydes were characterized by a homologous series of peak shifts due to the addition of multiple CH 2 units. This result is explained by the formation of high-molecular-weight (HMW) compounds that contain single or multiple aldehyde moieties. The HMW fragment peaks for the blank filters exposed to n-aldehydes were relatively weak, indicating an important contribution from the ambient aerosol components to the formation of the HMW compounds. Among the factors affecting the overall interaction of aldehydes with atmospheric aerosol components, gas phase diffusion possibly limited the reactions under the studied conditions; therefore, their occurrence to a similar degree in the atmosphere is not ruled out, at least for the reactions involving n-nonanal and decanal. The major formation pathways for the observed HMW products may be the self-reactions of n-aldehydes mediated by atmospheric aerosol components and the reactions of n-aldehydes with organic aerosol components. The observed formation of HMW compounds encourages further investigations into their effects on the aerosol properties as well as the organic aerosol mass in the atmosphere.

Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

In situ chitosan gelation initiated by atmospheric plasma treatment

This work reports on the feasibility of atmospheric dielectric barrier discharge (DBD) plasma as a novel synthetic pathway for the liquid phase gelation of chitosan. The DBD plasma chitosan gelation process did not significantly alter the chemical structure of the biopolymer as confirmed by FTIR study. However, the oxidation processes and local heating effect associated with the solvent evaporation during the plasma treatment could provoke both reaction of chitosan degradation and the cleavage of β-1–4-glycosidic linkages with the concomitant generation of aldehyde groups able to crosslink via Schiff-base with amino groups from other chitosan molecules. Shear viscosity measurements suggested the formation of chitosan fragments of lower molecular weight after the plasma treatment of 1% (w/v) chitosan and fragments of higher molecular weight after the plasma treatment of 2% (w/v) chitosan. The crosslinking density of hydrogels generated during the in situ DBD plasma chitosan gelation process increased as a function of the treatment time and concentration of chitosan. As of consequence of the increase of the cross-linking density, the equilibrium swelling ratio and water content decreased significantly.

A Complex of Equine Lysozyme and Oleic Acid with Bactericidal Activity against Streptococcus pneumoniae

HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET’s bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA’s bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments – an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes’ bactericidal activities, while the proteins are required both to solubilize and/or present the lipid at the bacterial membrane and likely to confer other and separate functions during the bacterial death.

Optimized enzymatic dual functions of PaPrx protein by proton irradiation

We investigated the effects of proton irradiation on the function and structure of the Pseudomonas aeruginosa peroxiredoxin (PaPrx). Polyacrylamide gel demonstrated that PaPrx proteins exposed to proton irradiation at several doses exhibited simultaneous formation of high molecular weight (HMW) complexes and fragmentation. Size-exclusion chromatography (SEC) analysis revealed that the number of fragments and very low molecular weight (LMW) structures increased as the proton irradiation dose increased. The peroxidase activity of irradiated PaPrx was preserved, and its chaperone activity was significantly increased by increasing the proton irradiation dose. The chaperone activity increased about 3–4 fold after 2.5 kGy proton irradiation, compared with that of non-irradiated PaPrx, and increased to almost the maximum activity after 10 kGy proton irradiation. We previously obtained functional switching in PaPrx proteins, by using gamma rays and electron beams as radiation sources, and found that the proteins exhibited increased chaperone activity but decreased peroxidase activity. Interestingly, in this study we newly found that proton irradiation could enhance both peroxidase and chaperone activities. Therefore, we can suggest proton irradiation as a novel protocol for conserved 2-Cys protein engineering.

Highly Sensitive GC/MS/MS Method for Quantitation of Amino and Nonamino Organic Acids

Metabolite profiling methods are important tools for measurement of metabolite pools in biological systems. While most metabolite profiling methods report relative intensities or depend on a few internal standards representing all metabolites, the ultimate requirement for a quantitative description of the metabolite pool in biological cells and fluids is absolute concentration determination. We report here a high-throughput and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) targeted metabolite profiling method enabling absolute quantification of all detected metabolites. The method is based on methyl chloroformate derivatization and quantification by spiking samples with metabolite standards separately derivatized with deuterated derivatization reagents. The traditional electron impact ionization is replaced with positive chemical ionization since the latter to a much larger extent preserve the molecular ion and other high molecular weight fragments. This made it easier to select unique MS/MS transitions among the many coeluting metabolites. Currently, the novel GC/MS/MS method comprises 67 common primary metabolites of which most belong to the groups of amino and nonamino organic acids. We show the applicability of the method on urine and serum samples. The method is a significant improvement of present methodology for quantitative GC/MS metabolite profiling of amino acids and nonamino organic acids.

EndoG Links Bnip3-Induced Mitochondrial Damage and Caspase-Independent DNA Fragmentation in Ischemic Cardiomyocytes

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

BackgroundApoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid.Methodology/Principal FindingsWe show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death.Conclusions/SignificanceOur results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

ARTEMIS Nuclease Facilitates Apoptotic Chromatin Cleavage

One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis.

Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups

The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20 kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal α-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.

Alterations in the desquamation-related proteolytic cleavage of corneodesmosin and other corneodesmosomal proteins in psoriatic lesional epidermis

Desquamation occurs after proteolysis of corneodesmosomal proteins, including corneodesmosin (CDSN), by proteases of the kallikrein family, particularly KLK7. Impaired desquamation is one of the features of psoriasis, and psoriasis-associated single nucleotide polymorphisms of the CDSN gene may potentially modify the proteolysis of the encoded protein. To test whether the proteolysis of CDSN and other corneodesmosomal components is altered in psoriatic epidermis. Total protein extracts obtained by tape-stripping of nonlesional and lesional skin from 11 patients were compared by immunoblotting experiments. An almost intact form of CDSN that has never been observed previously in the normal upper stratum corneum was detected in the lesional skin extracts, showing an altered proteolytic processing of the protein. This form was also observed in the nonlesional skin extracts, but in lower amounts. For most patients, increased amounts of desmoglein 1, plakoglobin and of high molecular weight fragments of desmocollin 1 were detected in the lesional skin. For most of them, similar amounts of KLK7 were immunodetected in both nonlesional and lesional skin extracts. No particular differences were observed related to the psoriasis type, the HLA-Cw6 status of the patients or any particular CDSN polymorphisms. We detected a near full-length form of CDSN that has not been previously observed in normal stratum corneum. The results suggest a reduced degradation of all corneodesmosomal proteins in psoriatic lesions which probably reflects the persistence of corneodesmosomes.

The role of cordycepin in cancer treatment via induction or inhibition of apoptosis: implication of polyadenylation in a cell type specific manner

Most anticancer drugs show their antiproliferative and cytotoxic activity via induction of apoptosis. In the present study we assessed the implication and role of cordycepin, a polyadenylation-specific inhibitor and a well-known chemotherapeutic drug, in apoptosis, induced by the anticancer drug etoposide. For this purpose, a variety of leukemia and lymphoma cell lines (U937, K562, HL-60, Daudi, Molt-4) were treated with the anticancer drugs etoposide and/or cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining, caspase-6 activity assay and DeltaPsi m reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. The results showed that PAP changes in all cell lines, in response to apoptosis induced by etoposide, in many cases even prior to hallmarks of apoptosis (endonucleosomal cleavage of DNA, DeltaPsi(m) reduction). A further elucidation to this apoptosis-polyadenylation correlation was added, by cell treatment with cordycepin, resulting in either suppression (U937, K562) or induction (HL-60) of the apoptotic process, according to the cell type. However, inhibition of polyadenylation did not influence the cell lines Daudi and Molt-4 used, where alternative apoptotic pathways are induced through cleavage of DNA into high molecular weight fragments.

Analytical pyrolysis as a tool for the characterization of natural organic matter—A comparison of different approaches

A substantial fraction of natural organic matter (NOM) consists of complex mixtures of macromolecular compounds. As a result, high-molecular weight fragments liberated during the pyrolysis of NOM often carry the most significant structural information. Their diagnostic value, however, is commonly limited by the preferential transfer of lower-boiling pyrolysis products to the GC column, compared to larger fragments. Non-discriminating pyrolysis is a technique that minimizes transfer losses of larger fragments, which is the problem with most conventional pyrolyzers. It allows a range of sample sizes from ∼15 mg for the study of samples of low organic carbon content to estimated sub-microgram sizes for bacterial biomass. In this study, the performance of the non-discriminating pyrolysis system in the characterization of natural organic matter (NOM) was compared to the performance of a conventional Pt-filament pyrolyzer. In addition, a more limited comparison was carried out with a Curie-point system. A variety of organic materials were selected to serve as NOM models: chitin, peat, and organic matter isolated from tertiary sands. Non-discriminating pyrolysis-GC/MS allowed for the observation of larger molecular fragments, such as traces of cholesterol in the pyrolysis of chitin. Also, the distribution of the homologous series such as that observed for alkyl benzenes was shifted towards higher molecular weight fragments with the non-discriminating system. Overall, the macromolecular structures within NOM were more pronounced when using non-discriminating pyrolysis, as shown by the higher abundance in the pyrograms of higher molecular weight fragments of various polyaromatic and long-chain aliphatic compounds. Characterization of NOM using non-discriminating Py-GC/MS reduces the ambiguity caused by mass discrimination against higher mass fragments compared to other pyrolysis approaches. This provides a better fingerprint of the organic material present in different sample types, including soils and sediments.

Getting the most out of fluorescent amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP™) is one of the most widely applied molecular marker detection systems used today. Among the reasons for its popularity are its reproducibility, capacity to generate large numbers of data points in a single assay, and “off-the-shelf” universal applicability. The original AFLP protocol was developed using radioactive detection. The transfer of this technique to fluorescent detection on automated DNA fragment analysers not only removed the undesirable requirement for radioactivity but also provided the possibility for increased effectiveness and detection throughput. Unfortunately, a number of problems are frequently encountered with fluorescent AFLPs, particularly failure to amplify high molecular-weight fragments and generation of nonuniform peak distributions. Here, we describe an improved generic protocol for fluorescent AFLPs achieved mainly thorough optimization of the multiplexed selective amplification reaction. This improved protocol gives increased production of valuable high molecular-weight markers and uniform peak intensities, facilitating unambiguous scoring. The protocol has been successfully applied, without further optimization, to species of Salix and Populus (Salicaceae), Melampsora (Melampsoraceae, rust fungi) and Heracleum (Apiaceae), as well as sugar beet ( Beta vulgaris L. subsp. vulgaris , Amaranthaceae), the endangered species Ranunculus kadzunensis Makino (Ranunculaceae), and to Aphidius ervi Haliday (Braconidae), a parasitoid wasp.

Human ovarian cancer ascites fluid contains a mixture of incompletely degraded soluble products of fibrin that collectively possess an antiangiogenic property

Ovarian cancer ascites fluid (OCAF) displayed an antiangiogenic property in a chick chorioallantoic membrane (CAM) assay. This property was attributed in part to angiostatin although angiostatin-free OCAF retained a net antiangiogenic property. Recently, immunopurified fibrin(ogen) degradation products (FDPs) from malignant effusions of VX2 tumor-burdened rabbits exhibited antiangiogenic activity on the CAM. We questioned whether the FDPs of OCAF were also antiangiogenic. FDPs were immunopurified from individual OCAF samples, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis /western blots, enzyme-linked immunosorbent assays, and CAM assays. FDPs of OCAFs consisted of soluble high molecular weight (MW) fragments (>200 kd; approximately 40% of total FDPs), D-dimer (approximately 180 kd; approximately 37%), fragment D (approximately 90 kd; approximately 15%), and fragment E (approximately 50 kd; approximately 8%); intact fibrinogen was absent. When applied to CAM surfaces (0.5-1.6 mg/10 mL), purified FDPs significantly reduced the area of chorionic capillaries from 90% (in controls) to 47% over a 48-h period; from CAM sections, capillary density was reduced from 60% (controls) to 26%. FDPs prepared from fibrinogen displayed a similar antiangiogenic effect. Further digestion of OCAF FDPs by human plasmin caused degradation of high MW fragments, releasing additional D-dimer, fragment D, and fragment E. Of the fibrinogen-related components, OCAF contained only soluble FDPs (including incompletely digested fibrin fragments). Collectively, these FDPs contributed to the net antiangiogenic property of ascites fluid.